Richard E. Myers

Comparison of Directly Measured Left Ventricular Wall Stress and Stress Calculated from Geometric Reference Figures

Mean left ventricular wall force was determined with a calibrated transmural auxotonic strain gauge in the left ventricle of six anesthetized, open-chest dogs with intact circulation. The gauge was oriented in the plane of the minor left ventricular equator, midway between the papillary muscles. Left ventricular internal volume was derived from the passive pressure-volume curve of the arrested heart and calculated mean wall stress was derived both from spherical and ellipsoidal reference figures for the left ventricle and compared with measured forces. Control left ventricular end-diastolic pressure averaged 3.0 ± 0.6 mm Hg (SE). At this level of end-diastolic pressure, measured peak wall stress averaged 97.2±14.4 g/cm2, whereas calculated peak wall stress averaged 79.3±9.9 and 118.6±12.9 g/cm2, for the spherical and ellipsoidal models, respectively. Measured end-diastolic wall force values averaged 9.4±4.5 and 29.2±8.1 g/cm2 at an end-diastolic pressure of 3.0 and 12.3 mm Hg, respectively. In all cases, stress values calculated from spherical reference figures for the left ventricle were significantly lower than those measured directly. In four other experiments, using right heart bypass, the ventricular septum was exposed and active wall force was determined at two or more sites on the left ventricular minor equator. Wall stress at these sites differed by an average of 15.3%, indicating that stresses around the minor equator are relatively uniform. These studies lend validity to the application of geometric models in the calculation of mean wall stress and favor the application of an ellipsoid for the geometric reference figure.

Adenylyl cyclase subtype-specific compartmentalization: differential regulation of L-type Ca2+ current in ventricular myocytes.

Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the &bgr;-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules. Objective: We take advantage of ACV and ACVI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes. Methods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for &bgr;1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current (ICa,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on ICa,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling. Conclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of ICa,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of &bgr;- adrenergic receptor and AC compartmentalization may also be important for &bgr;-adrenergic receptor signaling in other cell types.Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the β-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules. Objective: We take advantage of AC V and AC VI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes. Methods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for β1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current ( I Ca,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on I Ca,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling. Conclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of I Ca,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of β- adrenergic receptor and AC compartmentalization may also be important for β-adrenergic receptor signaling in other cell types. # Novelty and Significance {#article-title-41}

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.

Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl4)-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl4-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl4-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl4-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl4, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress.

Phorbol ester and endothelin-1 alter functional expression of Na+/Ca2+ exchange, K+, and Ca2+ currents in cultured neonatal rat myocytes

Endothelin-1 (ET-1) and activation of protein kinase C (PKC) have been implicated in alterations of myocyte function in cardiac hypertrophy and heart failure. Changes in cellular Ca2+ handling and electrophysiological properties also occur in these states and may contribute to mechanical dysfunction and arrhythmias. While ET-1 or PKC stimulation induces cellular hypertrophy in cultured neonatal rat ventricular myocytes (NRVMs), a system widely used in studies of hypertrophic signaling, there is little data about electrophysiological changes. Here we studied the effects of ET-1 (100 nM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 μM) on ionic currents in NRVMs. The acute effects of PMA or ET-1 (≤30 min) were small or insignificant. However, PMA or ET-1 exposure for 48-72 h increased cell capacitance by 100 or 25%, respectively, indicating cellular hypertrophy. ET-1 also slightly increased Ca2+ current density (T and L type). Na+/Ca2+ exchange current was increased by chronic pretreatment with either PMA or ET-1. In contrast, transient outward and delayed rectifier K+ currents were strongly downregulated by PMA or ET-1 pretreatment. Inward rectifier K+ current tended toward a decrease at larger negative potential, but time-independent outward K+ current was unaltered by either treatment. The enhanced inward and reduced outward currents also result in action potential prolongation after PMA or ET-1 pretreatment. We conclude that chronic PMA or ET-1 exposure in cultured NRVMs causes altered functional expression of cardiac ion currents, which mimic electrophysiological changes seen in whole animal and human hypertrophy and heart failure.

Feedback Mechanisms for Cardiac-Specific MicroRNAs and cAMP Signaling in Electrical Remodeling

Background—Loss of transient outward K+ current (Ito) is well documented in cardiac hypertrophy and failure both in animal models and in humans. Electrical remodeling contributes to prolonged action potential duration and increased incidence of arrhythmias. Furthermore, there is a growing body of evidence linking microRNA (miR) dysregulation to the progression of both conditions. In this study, we examined the mechanistic basis underlying miR dysregulation in electrical remodeling and revealed a novel interaction with the adrenergic signaling pathway. Methods and Results—We first used a tissue-specific knockout model of Dicer1 in cardiomyocytes to reveal the overall regulatory effect of miRs on the ionic currents and action potentials. We then validated the inducible cAMP early repressor as a target of miR-1 and took advantage of a clinically relevant model of post myocardial infarction and miR delivery to probe the mechanistic basis of miR dysregulation in electrical remodeling. These experiments revealed the role of inducible cAMP early repressor as a repressor of miR-1 and Ito, leading to prolonged action potential duration post myocardial infarction. In addition, delivery of miR-1 and miR-133a suppressed inducible cAMP early repressor expression and prevented both electrical remodeling and hypertrophy. Conclusions—Taken together, our results illuminate the mechanistic links between miRs, adrenergic signaling, and electrical remodeling. They also serve as a proof-of-concept for the therapeutic potential of miR delivery post myocardial infarction.

Adenylyl Cyclase Subtype–Specific Compartmentalization

Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the &bgr;-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules. Objective: We take advantage of ACV and ACVI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes. Methods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for &bgr;1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current (ICa,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on ICa,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling. Conclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of ICa,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of &bgr;- adrenergic receptor and AC compartmentalization may also be important for &bgr;-adrenergic receptor signaling in other cell types.Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the β-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules. Objective: We take advantage of AC V and AC VI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes. Methods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for β1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current ( I Ca,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on I Ca,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling. Conclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of I Ca,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of β- adrenergic receptor and AC compartmentalization may also be important for β-adrenergic receptor signaling in other cell types. # Novelty and Significance {#article-title-41}

Adenylyl Cyclase Subtype–Specific CompartmentalizationNovelty and Significance

Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the &bgr;-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules. Objective: We take advantage of ACV and ACVI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes. Methods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for &bgr;1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current (ICa,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on ICa,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling. Conclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of ICa,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of &bgr;- adrenergic receptor and AC compartmentalization may also be important for &bgr;-adrenergic receptor signaling in other cell types.Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the β-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules. Objective: We take advantage of AC V and AC VI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes. Methods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for β1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current ( I Ca,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on I Ca,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling. Conclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of I Ca,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of β- adrenergic receptor and AC compartmentalization may also be important for β-adrenergic receptor signaling in other cell types. # Novelty and Significance {#article-title-41}

Adenylyl Cyclase Subtype–Specific CompartmentalizationNovelty and Significance: Differential Regulation of L-Type Ca2+ Current in Ventricular Myocytes

Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the &bgr;-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules. Objective: We take advantage of ACV and ACVI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes. Methods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for &bgr;1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current (ICa,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on ICa,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling. Conclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of ICa,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of &bgr;- adrenergic receptor and AC compartmentalization may also be important for &bgr;-adrenergic receptor signaling in other cell types.Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the β-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules. Objective: We take advantage of AC V and AC VI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes. Methods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for β1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current ( I Ca,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on I Ca,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling. Conclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of I Ca,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of β- adrenergic receptor and AC compartmentalization may also be important for β-adrenergic receptor signaling in other cell types. # Novelty and Significance {#article-title-41}