Faculty Bibliography

BACKGROUND:Patients with chronic kidney disease (CKD) are at increased risk of developing cardiac arrhythmogenesis and sudden cardiac death; however, the basis for this association is incompletely known. METHODS:Here, using murine models of CKD, we examined interactions between kidney disease progression and structural, electrophysiological, and molecular cardiac remodeling. RESULTS:C57BL/6 mice with adenine supplemented in their diet developed progressive CKD. Electrocardiographically, CKD mice developed significant QT prolongation and episodes of bradycardia. Optical mapping of isolated-perfused hearts using voltage-sensitive dyes revealed significant prolongation of action potential duration with no change in epicardial conduction velocity. Patch-clamp studies of isolated ventricular cardiomyocytes revealed changes in sodium and potassium currents consistent with action potential duration prolongation. Global transcriptional profiling identified dysregulated expression of cellular stress response proteins RBM3 (RNA-binding motif protein 3) and CIRP (cold-inducible RNA-binding protein) that may underlay the ion channel remodeling. Unexpectedly, we found that female sex is a protective factor in the progression of CKD and its cardiac sequelae. CONCLUSIONS:Our data provide novel insights into the association between CKD and pathologic proarrhythmic cardiac remodeling. Cardiac cellular stress response pathways represent potential targets for pharmacologic intervention for CKD-induced heart rhythm disorders.

Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known on potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca2+ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels are activated during diastolic Ca2+ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mouse and pig. This activation involved Cx43 hemichannel Ca2+ entry and coupling to Ca2+ release microdomains at the intercalated disc resulting in enhanced Ca2+ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability as compared to non-failing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca2+ release is a novel, targetable, mechanism of cardiac arrhythmogenesis in heart failure.

Background: Elevated intracardiac pressure due to heart failure induces electrical and structural remodeling in the left atrium (LA) that begets atrial myopathy and arrhythmias. The underlying molecular pathways that drive atrial remodeling during cardiac pressure overload are poorly defined. The purpose of this study is to characterize the response of the ETV1 signaling axis in the LA during cardiac pressure overload in humans and mouse models and explore the role of ETV1 in atrial electrical and structural remodeling. Methods: We performed gene expression profiling in 265 left atrial samples from patients who underwent cardiac surgery. Comparative gene expression profiling was performed between two murine models of cardiac pressure overload, transverse aortic constriction (TAC) banding and Angiotensin II (AngII) infusion, and a genetic model of Etv1 cardiomyocyte-selective knockout (Etv1^f/fMlc2a^Cre/+). Results: Using the Cleveland Clinic biobank of human LA specimens, we found that ETV1 expression is decreased in patients with reduced ejection fraction. Consistent with its role as an important mediator of the Neuregulin-1 (NRG1) signaling pathway and activator of rapid conduction gene programming, we identified a direct correlation between ETV1 expression level and NRG1, ERBB4, SCN5A, and GJA5 levels in human LA samples. In a similar fashion to heart failure patients, we showed that left atrial ETV1 expression is downregulated at the RNA and protein levels in murine pressure overload models. Comparative analysis of LA RNA-seq datasets from TAC and AngII treated mice showed a high Pearson correlation, reflecting a highly ordered process by which the LA undergoes electrical and structural remodeling. Cardiac pressure overload produced a consistent downregulation of ErbB4, Etv1, Scn5a, and Gja5 and upregulation of profibrotic gene programming, which includes Tgfbr1/2, Igf1, and numerous collagen genes. Etv1^f/fMlc2a^Cre/+ mice displayed atrial conduction disease and arrhythmias. Correspondingly, the LA from Etv1^f/fMlc2a^Cre/+ mice showed downregulation of rapid conduction genes and upregulation of profibrotic gene programming, whereas analysis of a gain-of-function ETV1 RNA-seq dataset from neonatal rat ventricular myocytes transduced with Etv1 showed reciprocal changes. Conclusions: ETV1 is downregulated in the LA during cardiac pressure overload, contributing to both electrical and structural remodeling.

Objective: To explore the effect of real-time three-dimensional echocardiography (RT-3DE) combined with speckle tracking imaging (STI) for evaluation on the structure and function of left atrium (LA) in pregnant women.
Method(s): A total of 102 pregnant women underwent routine prenatal examinations were selected and examined by echocardiography twice in early pregnancy (8-12 weeks) and late pregnancy (32-36 weeks), respectively, and the data were enrolled into early pregnancy group and late pregnancy group. Meanwhile, 29 physical examinees in normal childbearing period were served as control group. RT-3DE was used to obtain LA maximum volume (Vmax), minimum volume (Vmin) and before atrial contraction volume (VpreA). LA total emptying fraction (TEF), expansion index (EI), active emptying fraction (AEF) and passive emptying fraction (PEF) were calculated. STI was used to obtain LA global longitudinal strain (GLS), which could be divided into reservoir phase (GLSr), conduit phase (GLScd) and contraction phase (GLSct). The above indexes were compared among three groups.
Result(s): The volume of LA increased with gestational age. The anteroposterior diameter, Vmax, Vmin and VpreA in early pregnancy group were higher than those in control group (all P<0.05). The anteroposterior diameter, Vmax, Vmin, VpreA, AEF and GLSct of late pregnancy group were higher, EI, PEF, GLSr and GLScd were lower than those in early pregnancy group and control group (all P<0.05), while there was no significant difference of TEF among groups(P>0.05).
Conclusion(s): The structure of LA began to change in the early pregnancy period, and both structure and function changed in the late pregnancy period. RT-3DE combined with STI could sensitively detect the compensatory regulation effect of LA on left ventricular diastolic function.
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Rationale: Fibroblast growth factor homologous factors (FHFs) are key regulators of sodium channel inactivation. Mutations in these critical proteins have been implicated in human diseases including Brugada syndrome, idiopathic ventricular arrhythmias, and epileptic encephalopathy. The underlying ionic mechanisms by which reduced sodium channel availability in Fhf2 knockout mice predisposes to abnormal excitability at the tissue level are not well defined. Objective: Using animal models and theoretical multicellular linear strands, we examined how FHF2 orchestrates the interdependency of sodium, calcium, and gap junctional conductances to safeguard cardiac conduction. Methods and Results:Fhf2^KO mice were challenged by reducing calcium conductance using verapamil or by reducing gap junctional conductance using carbenoxolone or by backcrossing into a connexin 43 heterozygous (Cx43^+/-) background. All conditions produced conduction block in Fhf2^KO mice, with Fhf2^WT showing normal impulse propagation. To explore the ionic mechanisms of block in Fhf2^KO hearts, multicellular linear strand models incorporating FHF2-deficient sodium channel inactivation properties were constructed and faithfully recapitulated conduction abnormalities seen in mutant hearts. The mechanisms of conduction block in mutant strands with reduced calcium conductance or gap junction uncoupling are very different. Enhanced sodium channel inactivation due to FHF2 deficiency shifts dependence onto calcium current to sustain electrotonic driving force, axial current flow, and action potential generation from cell-to-cell. In the setting of gap junction uncoupling, slower charging time from upstream cells conspires with accelerated sodium channel inactivation in mutant strands to prevent sufficient downstream cell charging for action potential propagation. Conclusions: FHF2-dependent effects on sodium channel inactivation ensure adequate sodium current reserve to safeguard against numerous threats to reliable cardiac impulse propagation.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction¹. A unique GPCR that is known to require heterodimerization for function^2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands^7,8, while GABAB2 couples with G proteins^9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail¹⁵. Although the VFT heterodimer structure has been resolved¹⁶, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.
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Background: Plakophilin-2 (PKP2) is classically defined as a protein of the desmosome, an intercellular adhesion structure that also acts as a signaling hub to maintain structural and electrical homeostasis. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events that occur consequent to its mutation. Here we sought to captureearly molecular/cellular events that can act as nascent substrates for subsequent arrhythmic/cardiomyopathic phenotypes.
Method(s): We used multiple quantitative imaging modalities, as well as biochemical and high-resolution mass spectrometry methods to study the functional/structural properties of cells/tissues derived from cardiomyocytespecific, tamoxifen-activated, PKP2 knockout mice ("PKP2cKO"). Studies were carried out 14 days post-tamoxifen injection, a time point preceding an overt electrical or structural phenotype.Myocytes from right or left ventricular free wall were studied separately, to detect functional/structural asymmetries.
Result(s): Most properties of PKP2cKO left ventricular (LV) myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+transients, increased frequency of spontaneous Ca2+release events, increased [Ca2+] in the cytoplasm and sarcoplasmic reticulum compartments, and dynamic Ca2+accumulation in mitochondria. In addition, RyR2 in RV presented enhanced sensitivity to Ca2+and preferential phosphorylation in a domain known to modulate Ca2+gating. RNAseq at 14 days post-TAM showed no relevant difference in transcript abundance between RV and LV, neither in control nor in PKP2cKO cells, suggesting that in the earliest stage, [Ca2+]i dysfunction is not transcriptional. Rather, we found an RV-predominant increase in membrane permeability that can permit Ca2+entry into the cell. Cx43 ablation mitigated the increase in membrane permeability, the accumulation of cytoplasmic Ca2+and the early stages of RV dysfunction.
Conclusion(s): Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy and that is, at least in part, mediated by a Cx43-dependent membrane conduit. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage

BACKGROUND:Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS:We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice ("PKP2cKO") 14 days post-tamoxifen (post-TAM) injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS:homeostasis. Similarly, PKC inhibition normalized spark frequency at comparable SR load levels. CONCLUSIONS:handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

As a long non-coding RNA (lncRNA) and a transcriptional regulator, Metastasis associated lung adenocarcioma transcript-1 (MALAT-1) has been reported to be associated with proliferation and metastasis of hepatocellular carcinoma (HCC). However, the effects of MALAT-1 single nucleotide polymorphisms (SNPs) on HCC remains poorly understood. This study, including 624 HCC cases and 618 controls, aimed to explore the potential associations between three common tagSNPs at MALAT-1 and HCC risk in a Southern Chinese population. No significant associations were observed between the three tagSNPs and HCC risk under any genetic models after adjusting for potential confounders. Additionally, there were no any significant associations in the stratified analysis, combined effect analysis, and multifactor dimensionality reduction (MDR) analysis. Unification analysis of mediation and interaction on HCC risk further showed that four decomposition of total effects ((controlled direct effect (CDE), the reference interaction effect (INTref), the mediated interaction effect (INTmed), or the pure indirect effect (PIE)) were also not significant. Neither was the association between the MALAT-1 SNPs and progression factors of HCC, including TNM staging, metastasis, and cancer embolus; Overall, this study suggested that tagSNPs rs11227209, rs619586, and rs3200401 at MALAT-1 were not significantly associated with HCC susceptibility. Nevertheless, large population-based studies are warranted to further explore the role of MALAT-1 SNPs in HCC incidence and development