Complex Arrhythmia Syndrome in a Knock-In Mouse Model Carrier of the N98S Calm1 Mutation.

BACKGROUND: Calmodulin mutations are associated with arrhythmia syndromes in humans. Exome sequencing previously identified a de novo mutation in CALM1 resulting in a p.N98S substitution in a patient with sinus bradycardia and stress-induced bidirectional ventricular ectopy. The objectives of the present study were to determine if mice carrying the N98S mutation knocked into Calm1 replicate the human arrhythmia phenotype and to examine arrhythmia mechanisms. METHODS: Mouse lines heterozygous for the Calm1N98S allele (Calm1N98S/+) were generated using CRISPR/Cas9 technology. Adult mutant mice and their wildtype littermates (Calm1+/+) underwent electrocardiographic monitoring. Ventricular de- and repolarization was assessed in isolated hearts using optical voltage mapping. Action potentials and whole-cell currents and [Ca2+]i, as well, were measured in single ventricular myocytes using the patch-clamp technique and fluorescence microscopy, respectively. The microelectrode technique was used for in situ membrane voltage monitoring of ventricular conduction fibers. RESULTS: Two biologically independent knock-in mouse lines heterozygous for the Calm1N98S allele were generated. Calm1N98S/+ mice of either sex and line exhibited sinus bradycardia, QTc interval prolongation, and catecholaminergic bidirectional ventricular tachycardia. Male mutant mice also showed QRS widening. Pharmacological blockade and activation of ß-adrenergic receptors rescued and exacerbated, respectively, the long-QT phenotype of Calm1N98S/+ mice. Optical and electric assessment of membrane potential in isolated hearts and single left ventricular myocytes, respectively, revealed ß-adrenergically induced delay of repolarization. ß-Adrenergic stimulation increased peak density, slowed inactivation, and left-shifted the activation curve of ICa.L significantly more in Calm1N98S/+ versus Calm1+/+ ventricular myocytes, increasing late ICa.L in the former. Rapidly paced Calm1N98S/+ ventricular myocytes showed increased propensity to delayed afterdepolarization-induced triggered activity, whereas in situ His-Purkinje fibers exhibited increased susceptibility for pause-dependent early afterdepolarizations. Epicardial mapping of Calm1N98S/+ hearts showed that both reentry and focal mechanisms contribute to arrhythmogenesis. CONCLUSIONS: Heterozygosity for the Calm1N98S mutation is causative of an arrhythmia syndrome characterized by sinus bradycardia, QRS widening, adrenergically mediated QTc interval prolongation, and bidirectional ventricular tachycardia. ß-Adrenergically induced ICa.L dysregulation contributes to the long-QT phenotype. Pause-dependent early afterdepolarizations and tachycardia-induced delayed afterdepolarizations originating in the His-Purkinje network and ventricular myocytes, respectively, constitute potential sources of arrhythmia in Calm1N98S/+ hearts.

Cardiac sympathetic innervation network shapes the myocardium by locally controlling cardiomyocyte size through the cellular proteolytic machinery.

KEY POINTS: The heart is innervated by a dense sympathetic neuron network which, in the short term, controls chronotropy and inotropy and, in the long term, regulates cardiomyocyte size. Acute neurogenic control of heart rate is achieved locally through direct neuro-cardiac coupling at specific junctional sites (neuro-cardiac junctions). The ventricular sympathetic network topology is well-defined and characteristic for each mammalian species. In the present study, we used cell size regulation to determine whether long-term modulation of cardiac structure is achieved via direct sympatho-cardiac coupling. Local density of cardiac innervation correlated with cell size throughout the myocardial walls in all mammalian species analysed, including humans. The data obtained suggest that constitutive neurogenic control of cardiomyocyte trophism occurs through direct intercellular signalling at neuro-cardiac junctions. ABSTRACT: It is widely appreciated that sympathetic stimulation of the heart involves a sharp increase in beating rate and significant enhancement of contractility. We have previously shown that, in addition to these evident functions, sympathetic neurons (SNs) also provide trophic input to cardiomyocytes (CMs), regulating cell and organ size. More recently, we have demonstrated that cardiac neurons establish direct interactions with CMs, allowing neuro-cardiac communication to occur locally, with a 'quasi-synaptic' mechanism. Based on the evidence that cardiac SNs are unevenly distributed throughout the myocardial walls, we investigated the hypothesis that CM size distribution reflects the topology of neuronal density. In vitro analyses of SN/CM co-cultures, ex vivo confocal and multiphoton imaging in clarified hearts, and biochemical and molecular approaches were employed, in both rodent and human heart biopsies. In line with the trophic effect of SNs, and with local neuro-cardiac communication, CMs, directly contacted by SNs in co-cultures, were larger than the non-targeted ones. This property reflects the distribution of CM size throughout the ventricles of intact mouse heart, in which cells in the outer myocardial layers, which were contacted by more neuronal processes, were larger than those in the less innervated subendocardial region. Such differences disappeared upon genetic or pharmacological interference with the trophic SN/CM signalling axis. Remarkably, CM size followed the SN distribution pattern in other mammals, including humans. Our data suggest that both the acute and chronic influence of SNs on cardiac function and structure is enacted as a result of the establishment of specific intercellular neuro-cardiac junctions.

Atrial fibrillation and electrophysiology in transgenic mice with cardiac-restricted overexpression of FKBP12.

Cardiomyocyte-restricted overexpression of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice develop spontaneous atrial fibrillation (AF). The aim of the present study is to explore the mechanisms underlying the occurrence of AF in αMyHC-FKBP12 mice. Spontaneous AF was documented by telemetry in vivo and Langendorff-perfused hearts of αMyHC-FKBP12 and littermate control mice in vitro. Atrial conduction velocity was evaluated by optical mapping. The patch-clamp technique was applied to determine the potentially altered electrophysiology in atrial myocytes. Channel protein expression levels were evaluated by Western blot analyses. Spontaneous AF was recorded in four of seven αMyHC-FKBP12 mice but in none of eight nontransgenic (NTG) controls. Atrial conduction velocity was significantly reduced in αMyHC-FKBP12 hearts compared with NTG hearts. Interestingly, the mean action potential duration at 50% but not 90% was significantly prolonged in αMyHC-FKBP12 atrial myocytes compared with their NTG counterparts. Consistent with decreased conduction velocity, average peak Na+ current ( INa) density was dramatically reduced and the INa inactivation curve was shifted by approximately +7 mV in αMyHC-FKBP12 atrial myocytes, whereas the activation and recovery curves were unaltered. The Nav1.5 expression level was significantly reduced in αMyHC-FKBP12 atria. Furthermore, we found increases in atrial Cav1.2 protein levels and peak L-type Ca2+ current density and increased levels of fibrosis in αMyHC-FKBP12 atria. In summary, cardiomyocyte-restricted overexpression of FKBP12 reduces the atrial Nav1.5 expression level and mean peak INa, which is associated with increased peak L-type Ca2+ current and interstitial fibrosis in atria. The combined electrophysiological and structural changes facilitated the development of local conduction block and altered action potential duration and spontaneous AF. NEW & NOTEWORTHY This study addresses a long-standing riddle regarding the role of FK506-binding protein 12 in cardiac physiology. The work provides further evidence that FK506-binding protein 12 is a critical component for regulating voltage-gated sodium current and in so doing has an important role in arrhythmogenic physiology, such as atrial fibrillation.

Ondansetron blocks wild-type and p.F503L variant small-conductance Ca2+-activated K+ channels.

Apamin-sensitive small-conductance Ca2+-activated K+ (SK) current ( IKAS) is encoded by Ca2+-activated K+ channel subfamily N ( KCNN) genes. IKAS importantly contributes to cardiac repolarization in conditions associated with reduced repolarization reserve. To test the hypothesis that IKAS inhibition contributes to drug-induced long QT syndrome (diLQTS), we screened for KCNN variants among patients with diLQTS, determined the properties of heterologously expressed wild-type (WT) and variant KCNN channels, and determined if the 5-HT3 receptor antagonist ondansetron blocks IKAS. We searched 2,306,335 records in the Indiana Network for Patient Care and found 11 patients with diLQTS who had DNA available in the Indiana Biobank. DNA sequencing discovered a heterozygous KCNN2 variant (p.F503L) in a 52-yr-old woman presenting with corrected QT interval prolongation at baseline (473 ms) and further corrected QT interval lengthening (601 ms) after oral administration of ondansetron. That patient was also heterozygous for the p.S38G and p.P2835S variants of the QT-controlling genes KCNE1 and ankyrin 2, respectively. Patch-clamp experiments revealed that the p.F503L KCNN2 variant heterologously expressed in human embryonic kidney (HEK)-293 cells augmented Ca2+ sensitivity, increasing IKAS density. The fraction of total F503L-KCNN2 protein retained in the membrane was higher than that of WT KCNN2 protein. Ondansetron at nanomolar concentrations inhibited WT and p.F503L SK2 channels expressed in HEK-293 cells as well as native SK channels in ventricular cardiomyocytes. Ondansetron-induced IKAS inhibition was also demonstrated in Langendorff-perfused murine hearts. In conclusion, the heterozygous p.F503L KCNN2 variant increases Ca2+ sensitivity and IKAS density in transfected HEK-293 cells. Ondansetron at therapeutic (i.e., nanomolar) concentrations is a potent IKAS blocker. NEW & NOTEWORTHY We showed that ondansetron, a 5-HT3 receptor antagonist, blocks small-conductance Ca2+-activated K+ (SK) current. Ondansetron may be useful in controlling arrhythmias in which increased SK current is a likely contributor. However, its SK-blocking effects may also facilitate the development of drug-induced long QT syndrome.

Critical Roles of STAT3 in ß-Adrenergic Functions in the Heart.

BACKGROUND: ß-Adrenergic receptors (ßARs) play paradoxical roles in the heart. On one hand, ßARs augment cardiac performance to fulfill the physiological demands, but on the other hand, prolonged activations of ßARs exert deleterious effects that result in heart failure. The signal transducer and activator of transcription 3 (STAT3) plays a dynamic role in integrating multiple cytokine signaling pathways in a number of tissues. Altered activation of STAT3 has been observed in failing hearts in both human patients and animal models. Our objective is to determine the potential regulatory roles of STAT3 in cardiac ßAR-mediated signaling and function. METHODS AND RESULTS: We observed that STAT3 can be directly activated in cardiomyocytes by ß-adrenergic agonists. To follow up this finding, we analyzed ßAR function in cardiomyocyte-restricted STAT3 knockouts and discovered that the conditional loss of STAT3 in cardiomyocytes markedly reduced the cardiac contractile response to acute ßAR stimulation, and caused disengagement of calcium coupling and muscle contraction. Under chronic ß-adrenergic stimulation, Stat3cKO hearts exhibited pronounced cardiomyocyte hypertrophy, cell death, and subsequent cardiac fibrosis. Biochemical and genetic data supported that Gαs and Src kinases are required for ßAR-mediated activation of STAT3. Finally, we demonstrated that STAT3 transcriptionally regulates several key components of ßAR pathway, including ß1AR, protein kinase A, and T-type Ca(2+) channels. CONCLUSIONS: Our data demonstrate for the first time that STAT3 has a fundamental role in ßAR signaling and functions in the heart. STAT3 serves as a critical transcriptional regulator for ßAR-mediated cardiac stress adaption, pathological remodeling, and heart failure.

Small-Conductance Calcium-Activated Potassium Current Is Activated During Hypokalemia and Masks Short-Term Cardiac Memory Induced by Ventricular Pacing.

BACKGROUND: Hypokalemia increases the vulnerability to ventricular fibrillation. We hypothesize that the apamin-sensitive small-conductance calcium-activated potassium current (IKAS) is activated during hypokalemia and that IKAS blockade is proarrhythmic. METHODS AND RESULTS: Optical mapping was performed in 23 Langendorff-perfused rabbit ventricles with atrioventricular block and either right or left ventricular pacing during normokalemia or hypokalemia. Apamin prolonged the action potential duration (APD) measured to 80% repolarization (APD80) by 26 milliseconds (95% confidence interval [CI], 14-37) during normokalemia and by 54 milliseconds (95% CI, 40-68) during hypokalemia (P=0.01) at a 1000-millisecond pacing cycle length. In hypokalemic ventricles, apamin increased the maximal slope of APD restitution, the pacing cycle length threshold of APD alternans, the pacing cycle length for wave-break induction, and the area of spatially discordant APD alternans. Apamin significantly facilitated the induction of sustained ventricular fibrillation (from 3 of 9 hearts to 9 of 9 hearts; P=0.009). Short-term cardiac memory was assessed by the slope of APD80 versus activation time. The slope increased from 0.01 (95% CI, -0.09 to 0.12) at baseline to 0.34 (95% CI, 0.23-0.44) after apamin (P<0.001) during right ventricular pacing and from 0.07 (95% CI, -0.05 to 0.20) to 0.54 (95% CI, 0.06-1.03) after apamin infusion (P=0.045) during left ventricular pacing. Patch-clamp studies confirmed increased IKAS in isolated rabbit ventricular myocytes during hypokalemia (P=0.038). CONCLUSIONS: Hypokalemia activates IKAS to shorten APD and maintain repolarization reserve at late activation sites during ventricular pacing. IKAS blockade prominently lengthens the APD at late activation sites and facilitates ventricular fibrillation induction.

A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy.

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.

Loss of Hand2 in a population of Periostin lineage cells results in pronounced bradycardia and neonatal death.

The Periostin Cre (Postn-Cre) lineage includes endocardial and neural crest derived mesenchymal cells of the cardiac cushions, neural crest-derived components of the sympathetic and enteric nervous systems, and cardiac fibroblasts. In this study, we use the Postn-Cre transgenic allele to conditionally ablate Hand2 (H2CKO). We find that Postn-Cre H2CKOs die shortly after birth despite a lack of obvious cardiac structural defects. To ascertain the cause of death, we performed a detailed comparison of the Postn-Cre lineage and Hand2 expression at mid and late stages of embryonic development. Gene expression analyses demonstrate that Postn-Cre ablates Hand2 from the adrenal medulla as well as the sphenopalatine ganglia of the head. In both cases, Hand2 loss-of-function dramatically reduces expression of Dopamine Beta Hydroxylase (Dbh), a gene encoding a crucial catecholaminergic biosynthetic enzyme. Expression of the genes Tyrosine Hydroxylase (Th) and Phenylethanolamine N-methyltransferase (Pnmt), which also encode essential catecholaminergic enzymes, were severely reduced in postnatal adrenal glands. Electrocardiograms demonstrate that 3-day postnatal Postn-Cre H2CKO pups exhibit sinus bradycardia. In conjunction with the aforementioned gene expression analyses, these results strongly suggest that the observed postnatal lethality occurs due to a catecholamine deficiency and subsequent heart failure.

Challenges measuring cardiomyocyte renewal.

Interventions to effect therapeutic cardiomyocyte renewal have received considerable interest of late. Such interventions, if successful, could give rise to myocardial regeneration in diseased hearts. Regenerative interventions fall into two broad categories, namely approaches based on promoting renewal of pre-existing cardiomyocytes and approaches based on cardiomyogenic stem cell activity. The latter category can be further subdivided into approaches promoting differentiation of endogenous cardiomyogenic stem cells, approaches wherein cardiomyogenic stem cells are harvested, amplified or enriched ex vivo, and subsequently engrafted into the heart, and approaches wherein an exogenous stem cell is induced to differentiate in vitro, and the resulting cardiomyocytes are engrafted into the heart. There is disagreement in the literature regarding the degree to which cardiomyocyte renewal occurs in the normal and injured heart, the mechanism(s) by which this occurs, and the degree to which therapeutic interventions can enhance regenerative growth. This review discusses several caveats which are encountered when attempting to measure cardiomyocyte renewal in vivo which likely contribute, at least in part, to the disagreement regarding the levels at which this occurs in normal, injured and treated hearts. This article is part of a Special Issue entitled: Cardiomyocyte biology: Cardiac pathways of differentiation, metabolism and contraction.

Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis.

Dishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, plays an important role in regulating the actin cytoskeleton via mediation of linear actin assembly. Previous functional studies of Daam1 in lower species suggest its essential role in Drosophila trachea formation and Xenopus gastrulation. However, its in vivo physiological function in mammalian systems is largely unknown. We have generated Daam1-deficient mice via gene-trap technology and found that Daam1 is highly expressed in developing murine organs, including the heart. Daam1-deficient mice exhibit embryonic and neonatal lethality and suffer multiple cardiac defects, including ventricular noncompaction, double outlet right ventricles and ventricular septal defects. In vivo genetic rescue experiments further confirm that the lethality of Daam1-deficient mice results from the inherent cardiac abnormalities. In-depth analyses have revealed that Daam1 is important for regulating filamentous actin assembly and organization, and consequently for cytoskeletal function in cardiomyocytes, which contributes to proper heart morphogenesis. Daam1 is also found to be important for proper cytoskeletal architecture and functionalities in embryonic fibroblasts. Biochemical analyses indicate that Daam1 does not regulate cytoskeletal organization through RhoA, Rac1 or Cdc42. Our study highlights a crucial role for Daam1 in regulating the actin cytoskeleton and tissue morphogenesis.

A Phox2- and Hand2-dependent Hand1 cis-regulatory element reveals a unique gene dosage requirement for Hand2 during sympathetic neurogenesis.

Neural crest cell specification and differentiation to a sympathetic neuronal fate serves as an important model for neurogenesis and depends upon the function of both bHLH transcription factors, notably Hand2, and homeodomain transcription factors, including Phox2b. Here, we define a 1007 bp cis-regulatory element 5' of the Hand1 gene sufficient to drive reporter expression within the sympathetic chain of transgenic mice. Comparative genomic analyses uncovered evolutionarily conserved consensus-binding sites within this element, which chromatin immunoprecipitation and electrophoretic mobility shift assays confirm are bound by Hand2 and Phox2b. Mutational analyses revealed that the conserved Phox2 and E-box binding sites are necessary for proper cis-regulatory element activity, and expression analyses on both Hand2 conditionally null and hypomorphic backgrounds demonstrate that Hand2 is required for reporter activation in a gene dosage-dependent manner. We demonstrate that Hand2 and Hand1 differentially bind the E-boxes in this cis-regulatory element, establishing molecular differences between these two factors. Finally, we demonstrate that Hand1 is dispensable for normal tyrosine hydroxylase (TH) and dopamine ß-hydroxylase (DBH) expression in sympathetic neurons, even when Hand2 gene dosage is concurrently reduced by half. Together, these data define a tissue-specific Hand1 cis-regulatory element controlled by two factors essential for the development of the sympathetic nervous system and provide in vivo regulatory evidence to support previous findings that Hand2, rather than Hand1, is predominantly responsible for regulating TH, DBH, and Hand1 expression in developing sympathetic neurons.

FKBP12 is a critical regulator of the heart rhythm and the cardiac voltage-gated sodium current in mice.

RATIONALE: FK506 binding protein (FKBP)12 is a known cis-trans peptidyl prolyl isomerase and highly expressed in the heart. Its role in regulating postnatal cardiac function remains largely unknown. METHODS AND RESULTS: We generated FKBP12 overexpressing transgenic (αMyHC-FKBP12) mice and cardiomyocyte-restricted FKBP12 conditional knockout (FKBP12(f/f)/αMyHC-Cre) mice and analyzed their cardiac electrophysiology in vivo and in vitro. A high incidence (38%) of sudden death was found in αMyHC-FKBP12 mice. Surface and ambulatory ECGs documented cardiac conduction defects, which were further confirmed by electric measurements and optical mapping in Langendorff-perfused hearts. αMyHC-FKBP12 hearts had slower action potential upstrokes and longer action potential durations. Whole-cell patch-clamp analyses demonstrated an ≈ 80% reduction in peak density of the tetrodotoxin-resistant, voltage-gated sodium current I(Na) in αMyHC-FKBP12 ventricular cardiomyocytes, a slower recovery of I(Na) from inactivation, shifts of steady-state activation and inactivation curves of I(Na) to more depolarized potentials, and augmentation of late I(Na), suggesting that the arrhythmogenic phenotype of αMyHC-FKBP12 mice is attributable to abnormal I(Na). Ventricular cardiomyocytes isolated from FKBP12(f/f)/αMyHC-Cre hearts showed faster action potential upstrokes and a more than 2-fold increase in peak I(Na) density. Dialysis of exogenous recombinant FKBP12 protein into FKBP12-deficient cardiomyocytes promptly recapitulated alterations in I(Na) seen in αMyHC-FKBP12 myocytes. CONCLUSIONS: FKBP12 is a critical regulator of I(Na) and is important for cardiac arrhythmogenic physiology. FKPB12-mediated dysregulation of I(Na) may underlie clinical arrhythmias associated with FK506 administration.

Interrogating functional integration between injected pluripotent stem cell-derived cells and surrogate cardiac tissue.

Myocardial infarction resulting in irreversible loss of cardiomyocytes (CMs) remains a leading cause of heart failure. Although cell transplantation has modestly improved cardiac function, major challenges including increasing cell survival, engraftment, and functional integration with host tissue, remain. Embryonic stem cells (ESCs), which can be differentiated into cardiac progenitors (CPs) and CMs, represent a candidate cell source for cardiac cell therapy. However, it is not known what specific cell type or condition is the most appropriate for transplantation. This problem is exasperated by the lack of efficient and predictive strategies to screen the large numbers of parameters that may impact cell transplantation. We used a cardiac tissue model, engineered heart tissue (EHT), and quantitative molecular and electrophysiological analyses, to test transplantation conditions and specific cell populations for their potential to functionally integrate with the host tissue. In this study, we validated our analytical platform using contractile mouse neonatal CMs (nCMs) and noncontractile cardiac fibroblasts (cFBs), and screened for the integration potential of ESC-derived CMs and CPs (ESC-CMs and -CPs). Consistent with previous in vivo studies, cFB injection interfered with electrical signal propagation, whereas injected nCMs improved tissue function. Purified bioreactor-generated ESC-CMs exhibited a diminished capacity for electrophysiological integration; a result correlated with lower (compared with nCMs) connexin 43 expression. ESC-CPs, however, appeared able to appropriately mature and integrate into EHT, enhancing the amplitude of tissue contraction. Our results support the use of EHT as a model system to accelerate development of cardiac cell therapy strategies.

Spatial variability in T-tubule and electrical remodeling of left ventricular epicardium in mouse hearts with transgenic Gαq overexpression-induced pathological hypertrophy.

Pathological left ventricular hypertrophy (LVH) is consistently associated with prolongation of the ventricular action potentials. A number of previous studies, employing various experimental models of hypertrophy, have revealed marked differences in the effects of hypertrophy on action potential duration (APD) between myocytes from endocardial and epicardial layers of the LV free wall. It is not known, however, whether pathological LVH is also accompanied by redistribution of APD among myocytes from the same layer in the LV free wall. In the experiments here, LV epicardial action potential remodeling was examined in a mouse model of decompensated LVH, produced by cardiac-restricted transgenic Gαq overexpression. Confocal linescanning-based optical recordings of propagated action potentials from individual in situ cardiomyocytes across the outer layer of the anterior LV epicardium demonstrated spatially non-uniform action potential prolongation in transgenic hearts, giving rise to alterations in spatial dispersion of epicardial repolarization. Local density and distribution of anti-Cx43 mmune reactivity in Gαq hearts were unchanged compared to wild-type hearts, suggesting preservation of intercellular coupling. Confocal microscopy also revealed heterogeneous disorganization of T-tubules in epicardial cardiomyocytes in situ. These data provide evidence of the existence of significant electrical and structural heterogeneity within the LV epicardial layer of hearts with transgenic Gαq overexpression-induced hypertrophy, and further support the notion that a small portion of electrically well connected LV tissue can maintain dispersion of action potential duration through heterogeneity in the activities of sarcolemmal ionic currents that control repolarization. It remains to be examined whether other experimental models of pathological LVH, including pressure overload LVH, similarly exhibit alterations in T-tubule organization and/or dispersion of repolarization within distinct layers of LV myocardium.

Ontogeny of cardiac sympathetic innervation and its implications for cardiac disease.

The vertebrate heart is innervated by the sympathetic and parasympathetic components of the peripheral autonomic nervous system, which regulates its contractile rate and force. Understanding the mechanisms that control sympathetic neuronal growth, differentiation, and innervation of the heart may provide insight into the etiology of cardiac arrhythmogenesis. This review provides an overview of the cell signaling pathways and transcriptional effectors that regulate both the noradrenergic gene program during sympathetic neurogenesis and regional nerve density during cardiac innervation. Recent studies exploring transcriptional regulation of the bHLH transcription factor Hand1 in developing sympathetic neurons are explored, and how the Hand1 sympathetic neuron-specific cis-regulatory element may be used further to assess the contribution of altered sympathetic innervation to human cardiac disease is discussed.

2011 Riley Heart Center Symposium on cardiac development: development of the cardiac conduction system and arrhythmias.

This article gives an overview of the 2011 Riley Heart Center Symposium that was held in Indianapolis, IN, USA, 11-13 September, 2011.

Functional screening of intracardiac cell transplants using two-photon fluorescence microscopy.

Although the adult mammalian myocardium exhibits a limited ability to undergo regenerative growth, its intrinsic renewal rate is insufficient to compensate for myocyte loss during cardiac disease. Transplantation of donor cardiomyocytes or cardiomyogenic stem cells is considered a promising strategy for reconstitution of cardiac mass, provided the engrafted cells functionally integrate with host myocardium and actively contribute to its contractile force. The authors previously developed a two-photon fluorescence microscopy-based assay that allows in situ screening of donor cell function after intracardiac delivery of the cells. This report reviews the techniques of two-photon fluorescence microscopy and summarizes its application for quantifying the extent to which a variety of donor cell types stably and functionally couple with the recipient myocardium.

The role of FK506-binding proteins 12 and 12.6 in regulating cardiac function.

Specifically, FK506-binding proteins 12 (FKBP12) and 12.6 (FKBP12.6) are cis-trans peptidyl prolyl isomerases that are expressed in the heart. Both FKBP12 and FKBP12.6 were previously known to interact with ryanodine receptors in striated muscles. Although FKBP12 is abundantly present in the heart, its function in the heart is largely uncertain. Recently, by generating FKBP12 transgenic overexpression and cardiac-restricted knockout mice, we showed that FKBP12 is critically important in regulating trans-sarcolemmal ionic currents, predominately the voltage-gated Na+ current, I(Na), but it appears to be less important for regulating cardiac ryanodine receptor function. Similar genetic approaches also confirm the role of FKBP12.6 in regulating cardiac ryanodine receptors. The current study demonstrated that FKBP12 and FKBP12.6 have very different physiologic functions in the heart.

Neonatal Deletion of Hand1 and Hand2 within Murine Cardiac Conduction System Reveals a Novel Role for HAND2 in Rhythm Homeostasis.

The cardiac conduction system, a network of specialized cells, is required for the functioning of the heart. The basic helix loop helix factors Hand1 and Hand2 are required for cardiac morphogenesis and have been implicated in cardiac conduction system development and maintenance. Here we use embryonic and post-natal specific Cre lines to interrogate the role of Hand1 and Hand2 in the function of the murine cardiac conduction system. Results demonstrate that loss of HAND1 in the post-natal conduction system does not result in any change in electrocardiogram parameters or within the ventricular conduction system as determined by optical voltage mapping. Deletion of Hand2 within the post-natal conduction system results in sex-dependent reduction in PR interval duration in these mice, suggesting a novel role for HAND2 in regulating the atrioventricular conduction. Surprisingly, results show that loss of both HAND factors within the post-natal conduction system does not cause any consistent changes in cardiac conduction system function. Deletion of Hand2 in the embryonic left ventricle results in inconsistent prolongation of PR interval and susceptibility to atrial arrhythmias. Thus, these results suggest a novel role for HAND2 in homeostasis of the murine cardiac conduction system and that HAND1 loss potentially rescues the shortened HAND2 PR phenotype.