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Healthy individuals exhibit blood pressure variation over a 24-hour period with higher blood pressure during wakefulness and lower blood pressure during sleep. Loss or disruption of the blood pressure circadian rhythm has been linked to adverse health outcomes, for example, cardiovascular disease, dementia, and chronic kidney disease. However, the current diagnostic and therapeutic approaches lack sufficient attention to the circadian rhythmicity of blood pressure. Sleep patterns, hormone release, eating habits, digestion, body temperature, renal and cardiovascular function, and other important host functions as well as gut microbiota exhibit circadian rhythms, and influence circadian rhythms of blood pressure. Potential benefits of nonpharmacologic interventions such as meal timing, and pharmacologic chronotherapeutic interventions, such as the bedtime administration of antihypertensive medications, have recently been suggested in some studies. However, the mechanisms underlying circadian rhythm-mediated blood pressure regulation and the efficacy of chronotherapy in hypertension remain unclear. This review summarizes the results of the National Heart, Lung, and Blood Institute workshop convened on October 27 to 29, 2021 to assess knowledge gaps and research opportunities in the study of circadian rhythm of blood pressure and chronotherapy for hypertension.

Sudden cardiac death (SCD) is the sudden, unexpected death due to abrupt loss of heart function secondary to cardiovascular disease. In certain populations living with cardiovascular disease, SCD follows a distinct 24-hour pattern in occurrence, suggesting day/night rhythms in behavior, the environment, and endogenous circadian rhythms result in daily spans of increased vulnerability. The National Heart, Lung, and Blood Institute convened a workshop, Understanding Circadian Mechanisms of Sudden Cardiac Death to identify fundamental questions regarding the role of the circadian rhythms in SCD. Part 2 summarizes research gaps and opportunities in the areas of population and clinical research identified in the workshop. Established research supports a complex interaction between circadian rhythms and physiological responses that increase the risk for SCD. Moreover, these physiological responses themselves are influenced by several biological variables, including the type of cardiovascular disease, sex, age, and genetics, as well as environmental factors. The emergence of new noninvasive biotechnological tools that continuously measure key cardiovascular variables, as well as the identification of biomarkers to assess circadian rhythms, hold promise for generating large-scale human data sets that will delineate which subsets of individuals are most vulnerable to SCD. Additionally, these data will improve our understanding of how people who suffer from circadian disruptions develop cardiovascular diseases that increase the risk for SCD. Emerging strategies to identify new biomarkers that can quantify circadian health (eg, environmental, behavioral, and internal misalignment) may lead to new interventions and therapeutic targets to prevent the progression of cardiovascular diseases that cause SCD.

Sudden cardiac death (SCD), the unexpected death due to acquired or genetic cardiovascular disease, follows distinct 24-hour patterns in occurrence. These 24-hour patterns likely reflect daily changes in arrhythmogenic triggers and the myocardial substrate caused by day/night rhythms in behavior, the environment, and endogenous circadian mechanisms. To better address fundamental questions regarding the circadian mechanisms, the National Heart, Lung, and Blood Institute convened a workshop, Understanding Circadian Mechanisms of Sudden Cardiac Death. We present a 2-part report of findings from this workshop. Part 1 summarizes the workshop and serves to identify research gaps and opportunities in the areas of basic and translational research. Among the gaps was the lack of standardization in animal studies for reporting environmental conditions (eg, timing of experiments relative to the light dark cycle or animal housing temperatures) that can impair rigor and reproducibility. Workshop participants also pointed to uncertainty regarding the importance of maintaining normal circadian rhythmic synchrony and the potential pathological impact of desynchrony on SCD risk. One related question raised was whether circadian mechanisms can be targeted to reduce SCD risk. Finally, the experts underscored the need for studies aimed at determining the physiological importance of circadian clocks in the many different cell types important to normal heart function and SCD. Addressing these gaps could lead to new therapeutic approaches/molecular targets that can mitigate the risk of SCD not only at certain times but over the entire 24-hour period.

Background Lamin A and C are nuclear filament proteins encoded by the LMNA gene. Mutations in the LMNA gene cause many congenital diseases known as laminopathies, including Emery–Dreifuss muscular dystrophy, Hutchinson–Gilford progeria syndrome, and familial dilated cardiomyopathy (DCM) with conduction disease. A missense mutation (N195K) in the A-type lamins results in familial DCM and sudden arrhythmic death. Objective The purpose of this study was to investigate the ion current mechanism of arrhythmia and DCM caused by the LaminA-N195K variant. Methods A homozygous mouse line expressing the Lmna-N195K mutation ( Lmna N195K/N195K ) that exhibited arrhythmia, DCM, and sudden death was used. Using whole cell patch-clamp technique, we measured action potential duration (APD), Na + currents (I Na ) in ventricular myocytes isolated from Lmna N195K/N195K , and wild-type mice. Results Both peak and late I Na were significantly ( P Lmna N195K/N195K ventricular myocytes. Similarly, Lmna N195K/N195K ventricular myocytes exhibited significant ( P 50 ] and 90% [APD 90 ] repolarization) and triggered activity. Acute application of ranolazine inhibited late I Na , shortened APD, and abolished triggered activity in Lmna N195K/N195K ventricular myocytes. Conclusion Inhibition of late I Na may be an effective therapy in preventing arrhythmia in patients with LmnaN195K mutation–related DCM.

We previously reported that the cardiomyocyte-specific leucine-rich repeat containing protein (LRRC)10 has critical functions in the mammalian heart. In the present study, we tested the role of LRRC10 in the response of the heart to biomechanical stress by performing transverse aortic constriction on Lrrc10-null ( Lrrc10−/−) mice. Mild pressure overload induced severe cardiac dysfunction and ventricular dilation in Lrrc10−/−mice compared with control mice. In addition to dilation and cardiomyopathy, Lrrc10−/−mice showed a pronounced increase in heart weight with pressure overload stimulation and a more dramatic loss of cardiac ventricular performance, collectively suggesting that the absence of LRRC10 renders the heart more disease prone with greater hypertrophy and structural remodeling, although rates of cardiac fibrosis and myocyte dropout were not different from control mice. Lrrc10−/−cardiomyocytes also exhibited reduced contractility in response to β-adrenergic stimulation, consistent with loss of cardiac ventricular performance after pressure overload. We have previously shown that LRRC10 interacts with actin in the heart. Here, we show that His150of LRRC10 was required for an interaction with actin, and this interaction was reduced after pressure overload, suggesting an integral role for LRRC10 in the response of the heart to mechanical stress. Importantly, these experiments demonstrated that LRRC10 is required to maintain cardiac performance in response to pressure overload and suggest that dysregulated expression or mutation of LRRC10 may greatly sensitize human patients to more severe cardiac disease in conditions such as chronic hypertension or aortic stenosis.

Aims: Mutations in CAV3-encoding caveolin-3 (Cav3) have been implicated in type 9 long QT syndrome (LQT9) and sudden infant death syndrome (SIDS). When co-expressed with SCN5A-encoded cardiac sodium channels these mutations increased late sodium current (INa) but the mechanism was unclear. The present study was designed to address the mechanism by which the LQT9-causing mutant Cav3-F97C affects the function of caveolar SCN5A. Methods and results: HEK-293 cells expressing SCN5A and LQT9 mutation Cav3-F97C resulted in a 2-fold increase in late INa compared to Cav3-WT. This increase was reversed by the neural nitric oxide synthase (nNOS) inhibitor L-NMMA. Based on these findings, we hypothesized that an nNOS complex mediated the effect of Cav3 on SCN5A. A SCN5A macromolecular complex was established in HEK-293 cells by transiently expressing SCN5A, α1-syntrophin (SNTA1), nNOS, and Cav3. Compared with Cav3-WT, Cav3-F97C produced significantly larger peak INa amplitudes, and showed 3.3-fold increase in the late INa associated with increased S-nitrosylation of SCN5A. L-NMMA reversed both the Cav3-F97C induced increase in late and peak INa and decreased S-nitrosylation of SCN5A. Overexpression of Cav3-F97C in adult rat cardiomyocytes caused a significant increase in late INa compared to Cav3-WT, and prolonged the action potential duration (APD90) in a nNOS-dependent manner. Conclusions: Cav3 is identified as an important negative regulator for cardiac late INa via nNOS dependent direct S-nitrosylation of SCN5A. This provides a molecular mechanism for how Cav3 mutations increase late INa to cause LQT9. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”.

Caveolae are specialized membrane microdomains enriched in cholesterol and sphingolipids which are present in multiple cell types including cardiomyocytes. Along with the essential scaffolding protein caveolin-3, a number of different ion channels and transporters have been localized to caveolae in cardiac myocytes including L-type Ca2+ channels (Ca(v)1.2), Na+ channels (Na(v)1.5), pacemaker channels (HCN4), Na+/Ca2+ exchanger (NCX1) and others. Closely associated with these channels are specific macromolecular signaling complexes that provide highly localized regulation of the channels. Mutations in the caveolin-3 gene (CAV3) have been linked with the congenital long QT syndrome (LQT9), and mutations in caveolar-localized ion channels may contribute to other inherited arrhythmias. Changes in the caveolar microdomain in acquired heart disease may also lead to dysregulation and dysfunction of ion channels, altering the risk of arrhythmias in conditions such as heart failure. This review highlights the existing evidence identifying and characterizing ion channels localized to caveolae in cardiomyocytes and their role in arrhythmogenesis.

Caveolin-3 (Cav-3) plays a critical role in organizing signaling molecules and ion channels involved in cardiac conduction and metabolism. Mutations in Cav-3 are implicated in cardiac conduction abnormalities and myopathies. Additionally, cardiac-specific overexpression of Cav-3 (Cav-3 OE) is protective against ischemic and hypertensive injury, suggesting a potential role for Cav-3 in basal cardiac electrophysiology and metabolism involved in stress adaptation. We hypothesized that overexpression of Cav-3 may alter baseline cardiac conduction and metabolism. We examined: (1) ECG telemetry recordings at baseline and during pharmacological interventions, (2) ion channels involved in cardiac conduction with immunoblotting and computational modeling, and (3) baseline metabolism in Cav-3 OE and transgene-negative littermate control mice. Cav-3 OE mice had decreased heart rates, prolonged PR intervals, and shortened QTc intervals with no difference in activity compared to control mice. Dobutamine or propranolol did not cause significant changes between experimental groups in maximal (dobutamine) or minimal (propranolol) heart rate. Cav-3 OE mice had an overall lower chronotropic response to atropine. The expression of Kv1.4 and Kv4.3 channels, Nav1.5 channels, and connexin 43 were increased in Cav-3 OE mice. A computational model integrating the immunoblotting results indicated shortened action potential duration in Cav-3 OE mice linking the change in channel expression to the observed electrophysiology phenotype. Metabolic profiling showed no gross differences in VO2, VCO2, respiratory exchange ratio, heat generation, and feeding or drinking. In conclusion, Cav-3 OE mice have changes in ECG intervals, heart rates, and cardiac ion channel expression. These findings give novel mechanistic insights into previously reported Cav-3 dependent cardioprotection.

Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca(2+) cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca(2+) signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca(2+) current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy.

Background— Congenital long-QT syndrome (LQTS) is a primary arrhythmogenic syndrome stemming from perturbed cardiac repolarization. LQTS, which affects ≈1 in 3000 persons, is 1 of the most common causes of autopsy-negative sudden death in the young. Since the sentinel discovery of cardiac channel gene mutations in LQTS in 1995, hundreds of mutations in 8 LQTS susceptibility genes have been identified. All 8 LQTS genotypes represent primary cardiac channel defects (ie, ion channelopathy) except LQT4, which is a functional channelopathy because of mutations in ankyrin-B. Approximately 25% of LQTS remains unexplained pathogenetically. We have pursued a “final common pathway” hypothesis to elicit novel LQTS-susceptibility genes. With the recent observation that the LQT3-associated, SCN5A -encoded cardiac sodium channel localizes in caveolae, which are known membrane microdomains whose major component in the striated muscle is caveolin-3, we hypothesized that mutations in caveolin-3 may represent a novel pathogenetic mechanism for LQTS. Methods and Results— Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, we performed open reading frame/splice site mutational analysis on CAV3 in 905 unrelated patients referred for LQTS genetic testing. CAV3 mutations were engineered by site-directed mutagenesis and the molecular phenotype determined by transient heterologous expression into cell lines that stably express the cardiac sodium channel hNa v 1.5. We identified 4 novel mutations in CAV3 -encoded caveolin-3 that were absent in >1000 control alleles. Electrophysiological analysis of sodium current in HEK293 cells stably expressing hNa v 1.5 and transiently transfected with wild-type and mutant caveolin-3 demonstrated that mutant caveolin-3 results in a 2- to 3-fold increase in late sodium current compared with wild-type caveolin-3. Our observations are similar to the increased late sodium current associated with LQT3-associated SCN5A mutations. Conclusions— The present study reports the first CAV3 mutations in subjects with LQTS, and we provide functional data demonstrating a gain-of-function increase in late sodium current.

The mechanisms underlying the blunted contractile response to β-adrenergic receptor (β-AR) stimulation in heart failure (HF) are incompletely understood, especially with regard to β-AR subtype–specific regulation of L-type Ca 2+ channels. We evaluated the impact of HF induced by pacing tachycardia on β-AR regulation of L-type Ca 2+ channels in a canine model. To evaluate changes in the relative subcellular distribution of β-AR subtypes, left ventricular membranes enriched in surface sarcolemma and T-tubular sarcolemma were prepared. Radioligand binding using [ 125 I]cyanopindolol revealed that HF resulted in a comparable decrease in the density of β 1 -ARs in both surface and T-tubule sarcolemma (55±4%, n=7, P P 2 -AR density was observed. Whole-cell patch clamp studies demonstrated a markedly blunted increase in I Ca,L in response to saturating concentrations of the nonselective β-AR agonist isoproterenol (0.1 μmol/L) in failing myocytes compared with control (129±20%, n=11, versus 332±35%, n=7; P 1 -AR– and β 2 -AR–selective stimulation showed that the major component of the blunted response to nonselective β-AR stimulation in HF was caused by β 2 -AR activation, resulting in a pertussis toxin–sensitive, G i -mediated inhibition of the β 1 -AR–induced increase in I Ca,L . In conclusion, canine HF results in the following: (1) a uniform reduction in β 1 -AR density in surface and T-tubule membrane fractions without a change in β 2 -AR density; and (2) the emergence of distinct G i -coupling to β 2 -ARs resulting in accentuated antagonism of β 1 -AR–mediated stimulation of I Ca,L . These results have implications for optimizing the use of β-AR drugs in HF.

The molecular genetic basis for early onset dilated cardiomyopathy (DCM) is largely unknown. We have identified that Lrrc10 is a cardiac-specific gene regulated by Nkx2.5 and GATA4. Lrrc10-null (Lrrc10-/-) mice exhibit prenatal systolic dysfunction followed by early onset DCM after birth. However, mechanistic roles for LRRC10 in the regulation of cardiac pathophysiology are unknown. We hypothesize that LRRC10 expression is critical for myocyte contractility and proper responses to pressure overload. Various mouse models with pathological cardiac hypertrophy showed decreased levels of Lrrc10. To test roles of LRRC10 in response to biomechanical stress, transverse aortic constriction (TAC) was performed. Lrrc10-/- mice showed significant increases in left ventricular inner diameter dimensions and drastically reduced % fractional shortening in response to 4 weeks of TAC (13%) after 4 weeks, while no significant changes occurred in WT after TAC (26% F.S.). These data indicate that pressure overload greatly exacerbates systolic dysfunction in Lrrc10-/- vs WT hearts. Next, the role of LRRC10 in cardiac excitation contraction coupling was investigated. Myocyte contractility and calcium transients were studied using the video-edge detection system and fura 2-AM loading at basal levels and following perfusion with Isoproterenol (ISO, 10 nM) at 0.5 - 2 Hz pacing. Cell shortening was significantly reduced following perfusion with ISO in Lrrc10-/- myocytes (55% and 49% at 1 and 2 Hz, respectively) vs WT. Calcium transient analysis showed that there were no differences in peak amplitudes of [Ca2+]i between Lrrc10-/- and WT myocytes at baseline or with ISO treatment. Whole-cell voltage clamp experiments demonstrated a significant reduction in the L-type Ca2+ current (ICa,L) density in Lrrc10-/- myocytes (-2.5 ± 0.2 pA/pF) compared to WT myocytes (-6 ± 0.6 pA/pF). The inactivation of the ICa,L in Lrrc10-/- myocytes was significantly delayed. Further, co-immunoprecipitation showed that LRRC10 directly associated with Cav1.2 subunit of LTCC, which correlates well with the localization of LRRC10 to dyadic regions in adult cardiomyocytes by TEM. Therefore, LRRC10 is a novel DCM candidate gene essential for normal myocyte contractility via regulating ICa,L.

Ca2+ channel blockers have been an important part of the cardiovascular pharmacological armamentarium for more than 2 decades. These agents were developed as antianginals and antihypertensives, but their indications have expanded to include treatment of certain arrhythmias because of their atrioventricular (AV) nodal blocking properties. Interestingly, the development of these compounds largely preceded our knowledge of the molecular composition and detailed functional properties of voltage-gated Ca2+ channels. In fact, Ca2+ channel blockers were critical in defining the distinct class of voltage-dependent Ca2+ channels referred to as L-type Ca2+ channels, which can be found in cardiac myocytes, skeletal myocytes, vascular smooth muscle cells, neurons, and endocrine cells among other cells. Despite this broad distribution of L-type Ca2+ channels, Ca2+ channel blockers have proven useful agents because they exhibit pharmacological specificity for vascular smooth muscle and cardiac muscle. This specificity is attributable to a variety of factors including the voltage and use-dependent blocking properties of these agents, subtle differences in the sensitivity of distinct isoforms of L-type Ca2+ channels present in different tissues, and the tissue distribution of the drugs. In addition, different classes of Ca2+ channel blockers vary in their relative potency to block vascular smooth muscle Ca2+ channels (antihypertensive/antianginal properties) relative to their block of AV nodal Ca2+ channels (antiarrhythmic properties). Alas, the specificity is not perfect, and so these agents can bring with them undesired side effects including constipation, peripheral edema, dizziness, and headache. In addition, problems can arise from the overlap of vascular smooth muscle and cardiac blocking properties. For example, if one wants to control a rapid heart rate in a hypotensive patient with atrial fibrillation by blocking AV nodal Ca2+ channels, accompanying vasodilation from vascular smooth muscle blockade can be problematic. Alternatively, treating angina …

Caveolae are discrete microdomains in cardiomyocytes that localize many ion channels and signaling proteins to enable compartmentalized regulation of cardiac signaling and physiology. Caveolin-3 (Cav-3) is a muscle specific scaffolding protein integral to caveolae in cardiomyocytes. Previous studies have indicated reduced Cav-3 expression and caveolae in cardiac hypertrophy and heart failure. These forms of heart disease are associated with prolonged cardiac repolarization, which puts patients at an increased risk for arrhythmias and sudden cardiac death. Furthermore, mutations in the CAV3 gene are linked to congenital Long QT syndrome (LQTS9). We hypothesize that caveolae provide integrated regulation of cardiac repolarization by controlling the density and functional properties of multiple classes of ion channels. To determine the impact of Cav-3 expression levels on cardiac repolarization we generated a cardiac-specific conditional Cav-3 knockout (Cav-3−/−) mouse. Western blot and transmission electron microscopy analysis demonstrated reduced expression of Cav-3 (88%) and the absence of caveolae respectively in the ventricular myocytes of Cav-3−/− mice compared to wild type (WT) mice. The Cav-3−/− mice demonstrate a significant increase in the telemetry ECG QT interval. Action potentials durations (APD) measured in ventricular myocytes from Cav-3−/− mice were significantly increased, APD50:30±9 ms and APD90:87±15 ms compared to littermate WT mice (APD50:9±2 ms; APD90:18±6 ms). Furthermore, whole-cell voltage clamp experiments in isolated ventricular myocytes from the Cav-3−/− mice demonstrated changes in a number of ionic currents including reduced peak Ito (19.6±2.4 pA/pF compared to 11±1.4 pA/pF in WT cells) and significantly delayed inactivation of ICa,L. We conclude that loss of Cav-3 expression impacts the function of multiple types of ion channels causing delayed cardiac repolarization and so may be integrally linked to arrhythmias and sudden cardiac death.

Summary Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gαo and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L-AP4, a type III mGluR-selective agonist, enhances Ca2+ uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L-AP4-induced Ca2+ influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gαo protein is absent in melanocytes. However, forced expression of Gαo restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of Gi/Go proteins, did not affect basal or mGluR6-induced Ca2+ uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.

Background— Type 2 long QT syndrome involves mutations in the human ether a-go-go–related gene ( hERG or KCNH2 ). T421M, an S1 domain mutation in the Kv11.1 channel protein, was identified in a resuscitated patient. We assessed its biophysical, protein trafficking, and pharmacological mechanisms in adult rat ventricular myocytes. Methods and Results— Isolated adult rat ventricular myocytes were infected with wild-type (WT)–Kv11.1– and T421M-Kv11.1–expressing adenovirus and analyzed with the use of patch clamp, Western blot, and confocal imaging techniques. Expression of WT-Kv11.1 or T421M-Kv11.1 produced peak tail current ( I Kv11.1 ) of 8.78±1.18 and 1.91±0.22 pA/pF, respectively. Loss of mutant I Kv11.1 resulted from (1) a partially trafficking-deficient channel protein with reduced cell surface expression and (2) altered channel gating with a positive shift in the voltage dependence of activation and altered kinetics of activation and deactivation. Coexpression of WT+T421M-Kv11.1 resulted in heterotetrameric channels that remained partially trafficking deficient with only a minimal increase in peak I Kv11.1 density, whereas the voltage dependence of channel gating became WT-like. In the adult rat ventricular myocyte model, both WT-Kv11.1 and T421M-Kv11.1 channels responded to β-adrenergic stimulation by increasing I Kv11.1 . Conclusions— The T421M-Kv11.1 mutation caused a loss of I Kv11.1 through interactions of abnormal protein trafficking and channel gating. Furthermore, for coexpressed WT+T421M-Kv11.1 channels, different dominant-negative interactions govern protein trafficking and ion channel gating, and these are likely to be reflected in the clinical phenotype. Our results also show that WT and mutant Kv11.1 channels responded to β-adrenergic stimulation.

Low voltage activated T-type calcium channels (TTCC) play a pivotal role in the developing heart. Although the TTCC isoforms, Cav3.1 and Cav3.2, underlie cardiac TTCC current (ICa,T) and are expressed in atrial and ventricular myocytes during development, their expression and roles recede in the adult heart. However, previous studies have demonstrated the re-expression of ICa,T in pathological cardiac hypertrophy, suggesting that TTCCs contribute to the altered Ca2+ cycling and signaling in these pathological conditions. In addition, the reported altered expression of some Cavβ subunits (specifically β1 and β2 subunits) of the high voltage activated L-type calcium channels (LTCC) has been reported in hypertrophy and heart failure. We hypothesize that the altered expression of the Cavβ subunits in cardiac hypertrophy and heart failure results in the altered coupling and regulation of ICa,T. We found an increase in the Cavβ1 and Cavβ3 mRNA and Cavβ2 and Cavβ4 subunit isoform at the protein level in mouse ventricles in a transthoracic aortic constriction (TAC) induced pathological cardiac hypertrophy model. Whole-cell patch clamp electrophysiology using transiently transfected HEK293 cells revealed that co-expression of Cavβ1 or Cavβ2 with Cav3.2 channel isoform resulted in a significant increase in peak ICav3.2 and a rightward shift in the V1/2 of activation and significantly slower inactivation of ICav3.2. On the other hand, co-expression of Cavβ3 or Cavβ4 significantly reduced the peak ICav3.2. In contrast, co-expression of Cavβ isoforms did not alter ICav3.1. Furthermore, co-immunoprecipitation studies in transiently transfected HEK293 cells also demonstrated that the Cav3.2 channel separately co-immunoprecitated with anti-Cavβ1 or anti-Cavβ2 antibody. In conclusion, our data suggest that the Cavβ1 and Cavβ2 subunits of the LTCC may regulate ICav3.2 in cardiomyocytes during pathological cardiac hypertrophy.

Evaluation of: Wei S, Guo A, Chen B et al.: T-tubule remodeling during transition from hypertrophy to heart failure. Circ. Res. 107(4), 520–531 (2010). The highly organized transverse (T)-tubule membrane system in ventricular myocytes plays an integral role in excitation–contraction coupling, and disruption of this T-tubule network has been implicated in contractile dysfunction in heart failure. Wei and colleagues used advanced confocal imaging techniques to evaluate cardiomyocyte membrane structure in intact rat hearts following surgically induced pressure overload, and demonstrated a loss of integrity of the T-tubule system. Importantly, this T-tubule dropout was detected early in the disease process, at the stage of compensated hypertrophy before observable ventricular dysfunction, and then progressed in proportion to the severity of the systolic dysfunction. These findings provide a new insight into the critical transition from compensated hypertrophy to heart failure and suggests important future areas for investigation.

Endothelin-1 (ET-1) is an autocrine factor in the mammalian heart important in enhancing cardiac performance, protecting against myocardial ischemia, and initiating the development of cardiac hypertrophy. The ETA receptor is a seven-transmembrane G-protein-coupled receptor whose precise subcellular localization in cardiac muscle is unknown. Here we used fluorescein ET-1 and 125I-ET-1 to provide evidence for ET-1 receptors in cardiac transverse tubules (T-tubules). Moreover, the ETA receptor and downstream effector phospholipase C-beta 1 were co-localized within T-tubules using standard immunofluorescence techniques, and protein kinase C (PKC)-epsilon-enhanced green fluorescent protein bound reversibly to T-tubules upon activation. Localized photorelease of diacylglycerol further suggested compartmentation of PKC signaling, with release at the myocyte "surface" mimicking the negative inotropic effects of bath-applied PKC activators and "deep" release mimicking the positive inotropic effect of ET-1. The functional significance of T-tubular ET-1 receptors was further tested by rendering the T-tubule lumen inaccessible to bath-applied ET-1. Such "detubulated" cardiac myocytes showed no positive inotropic response to 20 nM ET-1, despite retaining both a nearly normal twitch response to field stimulation and a robust positive inotropic response to 20 nm isoproterenol. We propose that ET-1 enhances myocyte contractility by activating ETA receptor-phospholipase C-beta 1-PKC-epsilon signaling complexes preferentially localized in cardiac T-tubules. Compartmentation of ET-1 signaling complexes may explain the discordant effects of ET-1 versus bath applied PKC activators and may contribute to both the specificity and diversity of the cardiac actions of ET-1.

Voltage gated T-type Ca2+ channel Cav3.2 subunit, responsible for T-type Ca2+ current (ICa,T) is expressed in different tissues including the heart and participates in Ca2+ influx, hormonal secretion, pacemaker activity and arrhythmia. The Cav3.2 channels are reported to be up regulated and contribute to the altered Ca2+ signaling and pathogenesis of cardiac hypertrophy via the activation of calcineurin/nuclear factor of activated T cells (NFAT) pathway. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels and signaling proteins, and provide temporal and spatial regulation of intracellular Ca2+ in cardiomyocytes. However, the mechanism of altered Ca2+ signaling in cardiac hypertrophy is not clearly defined. We investigated the role of caveolae and Cav3.2 channels in the regulation of Ca2+ signaling during angiotensin-II induced cardiac hypertrophy in ventricular myocytes. Immunogold labeling and electron microscopy analysis demonstrated the co-localization of Cav3.2 channel and Cav-3 relative to caveolae in the ventricular myocytes. GST pull-down analysis confirmed that the N-terminus region of Cav-3 interacts with Cav3.2 channels. Impact of Cav-3 association with CaV3.2 was analyzed by whole cell patch clamp technique. Co-expression of Cav-3 specifically inhibited ICav3.2 in heterologously expressed HEK293 cells. In the neonatal ventricular myocytes, overexpression of Cav-3 inhibited ICa,T and specifically inhibited the adenovirus (AdCav3.2) mediated peak Cav3.2 currents. In addition, overexpression of Cav-3 prevented the angiotensin-II induced hypertrophic responses in neonatal mouse cardiomyocytes. Over expression of Cav-3 prevented the angiotensin-II induced translocation of NFAT4 to the nucleus and also inhibited the phosphorylation of extracellular signal-regulated kinase, ERK. Overall our results demonstrate that over expression of Cav-3 suppresses pathological hypertrophic responses in cardiomyocytes via the inhibition of the ICav3.2. We conclude that Cav-3 may play a crucial role in protective signaling mechanisms in the ventricular myocytes during hypertrophic cardiomyopathy.

Objectives We hypothesized that cardiac myocyte-specific overexpression of caveolin-3 (Cav-3), a muscle-specific caveolin, would alter natriuretic peptide signaling and attenuate cardiac hypertrophy. Background Natriuretic peptides modulate cardiac hypertrophy and are potential therapeutic options for patients with heart failure. Caveolae, microdomains in the plasma membrane that contain caveolin proteins and natriuretic peptide receptors, have been implicated in cardiac hypertrophy and natriuretic peptide localization. Methods We generated transgenic mice with cardiac myocyte-specific overexpression of caveolin-3 (Cav-3 OE) and also used an adenoviral construct to increase Cav-3 in cardiac myocytes. Results The Cav-3 OE mice subjected to transverse aortic constriction had increased survival, reduced cardiac hypertrophy, and maintenance of cardiac function compared with control mice. In left ventricle at baseline, messenger ribonucleic acid for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were increased 7- and 3-fold, respectively, in Cav-3 OE mice compared with control subjects and were accompanied by increased protein expression for ANP and BNP. In addition, ventricles from Cav-3 OE mice had greater cyclic guanosine monophosphate levels, less nuclear factor of activated T-cell nuclear translocation, and more nuclear Akt phosphorylation than ventricles from control subjects. Cardiac myocytes incubated with Cav-3 adenovirus showed increased expression of Cav-3, ANP, and Akt phosphorylation. Incubation with methyl-β-cyclodextrin, which disrupts caveolae, or with wortmannin, a PI3K inhibitor, blocked the increase in ANP expression. Conclusions These results imply that cardiac myocyte-specific Cav-3 OE is a novel strategy to enhance natriuretic peptide expression, attenuate hypertrophy, and possibly exploit the therapeutic benefits of natriuretic peptides in cardiac hypertrophy and heart failure.

Prolongation of the repolarization action potential duration (APD) and altered Ca2+ signaling are associated with aging related cardiac hypertrophy and contractile dysfunction. However the mechanism of age dependent alteration in calcium signaling and arrhythmia is not known. Caveolin 3 (Cav3), a muscle-specific scaffolding protein of caveolae, is known to regulate voltage-gated Ca2+ channels and Ca2+ signaling in cardiomyocytes. To investigate the role of Cav3 in the cardiac aging, we used 4 and 24-month old WT type mice and age matched cardiac restricted Cav3 overexpresser (Cav3OE) mice. The 24-months aged WT mice developed ventricular hypertrophy and fibrosis. A significant reduction in the ejection fraction and fractional shortening confirmed impaired cardiac function in 24-months aged mice. Western blot analysis showed reduced Cav3 expression (50%) in the ventricular myocytes of 24-months old WT mice as compared to 4-months adult mice. In contrast, cardiac specific Cav3OE mice had normal cardiac function with no detectable fibrosis and cardiac hypertrophy at 24-months, which was similar to 4-months adult WT or Cav3OE mice. Whole-cell patch clamp analysis showed significantly increased APD50 (44.89±8ms) and APD90 (66.5±11ms) (n=6) in the ventricular myocytes of 24-month aged mice compared to 4-months adult mice (APD50: 8.9±2ms; APD90: 17.88±6ms; n=6). A re-expression of the ICa,T (−2.1±1pA/pF) observed in ventricular myocytes from 24-months aged WT mice suggested possible cause for altered Ca2+ signaling and induction of ventricular hypertrophy. In contrast, ventricular myocytes from 24-months aged Cav3OE showed a normalized action potential (APD50:11±3ms; APD90:47.5±6ms; n=8) and negligible ICa,T (−0.3± 0.5 pA/pF). We conclude that a reduced Cav3 expression in aged ventricle contributes to arrhythmia, altered Ca2+ signaling via re-expressed ICa,T and cardiac dysfunction. Cardiac specific overexpression of Cav3 prevents aging induced arrhythmia, cardiac dysfunction and hypertrophy.

Voltage-gated T-type Ca2+ channel (TTCC), Cav3.1 and Cav3.2, are normally expressed during cardiac development but are re-expressed in cardiac hypertrophy and may contribute to the altered intracellular Ca2+ during this disease. However, the mechanism of altered Ca2+ signaling in cardiac hypertrophy is not clearly known. Caveolae containing scaffolding protein caveolin-3 (Cav-3), provide spatiotemporal regulation of intracellular Ca2+ in cardiomyocytes. To define the source of signaling Ca2+ involved and basis of dysregulated contractile function in cardiac hypertrophy and to investigate the role of caveolae and TTCC in the regulation of Ca2+ signaling, we used a transthorasic aortic constriction (TAC) induced mouse model of cardiac hypertrophy. Western blot analysis revealed re-expression of Cav3.1 and Cav3.2 proteins and significant increase in expression of Cav-3 in adult ventricle from TAC mice but not from sham treated mice. Electron microscopy analysis demonstrated significant increase in the number of caveolae and co-localization of Cav3.2 and Cav-3 in the ventricular myocytes in the TAC hearts. Co-immunoprecipitation analysis using anti-Cav-3 antibody revealed that re-expressed Cav3.2 co-IPs with Cav-3 in the TAC hearts, but not in sham hearts. GST pull-down analysis using Cav-3 fusion proteins confirmed that Cav-3 directly associates with Cav3.2 channels. Whole cell patch clamp analysis in HEK293 cells co-expressed with either CaV3.2 and wild-type Cav-3 or GFP revealed that co-expression of Cav3.2 + Cav-3 significantly decreased the peak ICav3.2 (−12 ± 3 pA/pF, n=11) compared to Cav3.2+GFP (−31 ± 4 pA/pF, n=11). Whereas co-expression of Cav-3 had no effect on the ICav3.1. Cav-3 coexpression did affect the voltage dependent activation or inactivation of ICav3.2. We conclude that Cav-3 associates with Cav3.2 channels and regulates its function. Increased Cav-3 expression may play a crucial role in regulation of Ca2+ signaling during hypertrophic cardiomyopathy.