Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

RATIONALE: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. OBJECTIVE: The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. METHODS AND RESULTS: RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor ß1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor ß1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor ß1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor ß1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice (Txndc5-/-) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 (P<0.001) in Txndc5-/- versus wild-type mice, respectively. CONCLUSIONS: The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against ß agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.

Differential Expression and Remodeling of Transient Outward Potassium Currents in Human Left Ventricles.

BACKGROUND: Myocardial, transient, outward currents, Ito, have been shown to play pivotal roles in action potential (AP) repolarization and remodeling in animal models. The properties and contribution of Ito to left ventricular (LV) repolarization in the human heart, however, are poorly defined. METHODS AND RESULTS: Whole-cell, voltage-clamp recordings, acquired at physiological (35°C to 37°C) temperatures, from myocytes isolated from the LV of nonfailing human hearts identified 2 distinct transient currents, Ito,fast (Ito,f) and Ito,slow (Ito,s), with significantly (P<0.0001) different rates of recovery from inactivation and pharmacological sensitives: Ito,f recovers in ≈10 ms, 100× faster than Ito,s, and is selectively blocked by the Kv4 channel toxin, SNX-482. Current-clamp experiments revealed regional differences in AP waveforms, notably a phase 1 notch in LV subepicardial myocytes. Dynamic clamp-mediated addition/removal of modeled human ventricular Ito,f, resulted in hyperpolarization or depolarization, respectively, of the notch potential, whereas slowing the rate of Ito,f inactivation resulted in AP collapse. AP-clamp experiments demonstrated that changes in notch potentials modified the time course and amplitudes of voltage-gated Ca2+ currents, ICa. In failing LV subepicardial myocytes, Ito,f was reduced and Ito,s was increased, notch and plateau potentials were depolarized (P<0.0001) and AP durations were prolonged (P<0.001). CONCLUSIONS: Ito,f and Ito,s are differentially expressed in nonfailing human LV, contributing to regional heterogeneities in AP waveforms. Ito,f regulates notch and plateau potentials and modulates the time course and amplitude of ICa. Slowing Ito,f inactivation results in dramatic AP shortening. Remodeling of Ito,f in failing human LV subepicardial myocytes attenuates transmural differences in AP waveforms.

Arrhythmogenic remodeling of ß2 versus ß1 adrenergic signaling in the human failing heart.

BACKGROUND: Arrhythmia is the major cause of death in patients with heart failure, for which ß-adrenergic receptor blockers are a mainstay therapy. But the role of ß-adrenergic signaling in electrophysiology and arrhythmias has never been studied in human ventricles. METHODS AND RESULTS: We used optical imaging of action potentials and [Ca(2+)]i transients to compare the ß1- and ß2-adrenergic responses in left ventricular wedge preparations of human donor and failing hearts. ß1-Stimulation significantly increased conduction velocity, shortened action potential duration, and [Ca(2+)]i transients duration (CaD) in donor but not in failing hearts, because of desensitization of ß1-adrenergic receptor in heart failure. In contrast, ß2-stimulation increased conduction velocity in both donor and failing hearts but shortened action potential duration only in failing hearts. ß2-Stimulation also affected transmural heterogeneity in action potential duration but not in [Ca(2+)]i transients duration. Both ß1- and ß2-stimulation augmented the vulnerability and frequency of ectopic activity and enhanced substrates for ventricular tachycardia in failing, but not in donor, hearts. Both ß1- and ß2-stimulation enhanced Purkinje fiber automaticity, whereas only ß2-stimulation promoted Ca-mediated premature ventricular contractions in heart failure. CONCLUSIONS: During end-stage heart failure, ß2-stimulation creates arrhythmogenic substrates via conduction velocity regulation and transmurally heterogeneous repolarization. ß2-Stimulation is, therefore, more arrhythmogenic than ß1-stimulation. In particular, ß2-stimulation increases the transmural difference between [Ca(2+)]i transients duration and action potential duration, which facilitates the formation of delayed afterdepolarizations.

Deep RNA sequencing reveals dynamic regulation of myocardial noncoding RNAs in failing human heart and remodeling with mechanical circulatory support.

BACKGROUND: Microarrays have been used extensively to profile transcriptome remodeling in failing human heart, although the genomic coverage provided is limited and fails to provide a detailed picture of the myocardial transcriptome landscape. Here, we describe sequencing-based transcriptome profiling, providing comprehensive analysis of myocardial mRNA, microRNA (miRNA), and long noncoding RNA (lncRNA) expression in failing human heart before and after mechanical support with a left ventricular (LV) assist device (LVAD). METHODS AND RESULTS: Deep sequencing of RNA isolated from paired nonischemic (NICM; n=8) and ischemic (ICM; n=8) human failing LV samples collected before and after LVAD and from nonfailing human LV (n=8) was conducted. These analyses revealed high abundance of mRNA (37%) and lncRNA (71%) of mitochondrial origin. miRNASeq revealed 160 and 147 differentially expressed miRNAs in ICM and NICM, respectively, compared with nonfailing LV. Among these, only 2 (ICM) and 5 (NICM) miRNAs are normalized with LVAD. RNASeq detected 18 480, including 113 novel, lncRNAs in human LV. Among the 679 (ICM) and 570 (NICM) lncRNAs differentially expressed with heart failure, ≈10% are improved or normalized with LVAD. In addition, the expression signature of lncRNAs, but not miRNAs or mRNAs, distinguishes ICM from NICM. Further analysis suggests that cis-gene regulation represents a major mechanism of action of human cardiac lncRNAs. CONCLUSIONS: The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

Heterogeneity and function of K(ATP) channels in canine hearts.

BACKGROUND: The concept that pore-forming Kir6.2 and regulatory SUR2A subunits form cardiac ATP-sensitive potassium (K(ATP)) channels is challenged by recent reports that SUR1 is predominant in mouse atrial K(ATP) channels. OBJECTIVE: To assess SUR subunit composition of K(ATP) channels and consequence of K(ATP) activation for action potential duration (APD) in dog hearts. METHODS: Patch-clamp techniques were used on isolated dog cardiomyocytes to investigate K(ATP) channel properties. Dynamic current clamp, by injection of a linear K(+) conductance to simulate activation of the native current, was used to study the consequences of K(ATP) activation on APD. RESULTS: Metabolic inhibitor (MI)-activated current was not significantly different from pinacidil (SUR2A-specific)-activated current, and both currents were larger than diazoxide (SUR1-specific)-activated current in both the atrium and the ventricle. Mean K(ATP) conductance (activated by MI) did not differ significantly between chambers, although, within the ventricle, both MI-induced and pinacidil-induced currents tended to decrease from the epicardium to the endocardium. Dynamic current-clamp results indicate that myocytes with longer baseline APDs are more susceptible to injected K(ATP) current, a result reproduced in silico by using a canine action potential model (Hund-Rudy) to simulate epicardial and endocardial myocytes. CONCLUSIONS: Even a small fraction of K(ATP) activation significantly shortens APD in a manner that depends on existing heterogeneity in K(ATP) current and APD.

How to build an integrated biobank: the Washington University Translational Cardiovascular Biobank & Repository experience.

Translational studies that assess and extend observations made in animal models of human pathology to elucidate relevant and important determinants of human diseases require the availability of viable human tissue samples. However, there are a number of technical and practical obstacles that must be overcome in order to perform cellular and electrophysiological studies of the human heart. In addition, changing paradigms of how diseases are diagnosed, studied and treated require increasingly complex integration of rigorous disease phenotyping, tissue characterization and detailed delineation of a multitude of "_omics". Realizing the need for quality-controlled human cardiovascular tissue acquisition, annotation, biobanking and distribution, we established the Translational Cardiovascular Biobank & Repository at Washington University School of Medicine. Several critical details are essential for the success of cardiovascular biobanking including coordinated, trained and dedicated staff members; adequate, nonrestrictive informed consent protocols; and fully integrated clinical data management applications for annotating, tracking and sharing of tissue and data resources. Labor and capital investments into growing biobanking resources will facilitate collaborative efforts aimed at limiting morbidity and mortality due to heart disease and improving overall cardiovascular health.

Gender differences in electrophysiological gene expression in failing and non-failing human hearts.

The increasing availability of human cardiac tissues for study are critically important in increasing our understanding of the impact of gender, age, and other parameters, such as medications and cardiac disease, on arrhythmia susceptibility. In this study, we aimed to compare the mRNA expression of 89 ion channel subunits, calcium handling proteins, and transcription factors important in cardiac conduction and arrhythmogenesis in the left atria (LA) and ventricles (LV) of failing and nonfailing human hearts of both genders. Total RNA samples, prepared from failing male (n = 9) and female (n = 7), and from nonfailing male (n = 9) and female (n = 9) hearts, were probed using custom-designed Taqman gene arrays. Analyses were performed to explore the relationships between gender, failure state, and chamber expression. Hierarchical cluster analysis revealed chamber specific expression patterns, but failed to identify disease- or gender-dependent clustering. Gender-specific analysis showed lower expression levels in transcripts encoding for K(v)4.3, KChIP2, K(v)1.5, and K(ir)3.1 in the failing female as compared with the male LA. Analysis of LV transcripts, however, did not reveal significant differences based on gender. Overall, our data highlight the differential expression and transcriptional remodeling of ion channel subunits in the human heart as a function of gender and cardiac disease. Furthermore, the availability of such data sets will allow for the development of disease-, gender-, and, most importantly, patient-specific cardiac models, with the ability to utilize such information as mRNA expression to predict cardiac phenotype.

CD36 protein influences myocardial Ca2+ homeostasis and phospholipid metabolism: conduction anomalies in CD36-deficient mice during fasting.

Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca(2+) flux and activation of Ca(2+)-dependent phospholipases A(2) that cycle polyunsaturated FA into phospholipids. It is unknown whether CD36 deficiency influences myocardial Ca(2+) handling and phospholipid metabolism, which could compromise the heart, typically during stresses. Myocardial function was examined in fed or fasted (18-22 h) CD36(-/-) and WT mice. Echocardiography and telemetry identified conduction anomalies that were associated with the incidence of sudden death in fasted CD36(-/-) mice. No anomalies or death occurred in WT mice during fasting. Optical imaging of perfused hearts from fasted CD36(-/-) mice documented prolongation of Ca(2+) transients. Consistent with this, knockdown of CD36 in cardiomyocytes delayed clearance of cytosolic Ca(2+). Hearts of CD36(-/-) mice (fed or fasted) had 3-fold higher SERCA2a and 40% lower phospholamban levels. Phospholamban phosphorylation by protein kinase A (PKA) was enhanced after fasting reflecting increased PKA activity and cAMP levels in CD36(-/-) hearts. Abnormal Ca(2+) homeostasis in the CD36(-/-) myocardium associated with increased lysophospholipid content and a higher proportion of 22:6 FA in phospholipids suggests altered phospholipase A(2) activity and changes in membrane dynamics. The data support the role of CD36 in coordinating Ca(2+) homeostasis and lipid metabolism and the importance of this role during myocardial adaptation to fasting. Potential relevance of the findings to CD36-deficient humans would need to be determined.

Differential calcium handling in two canine models of right ventricular pressure overload.

BACKGROUND: The purpose of this investigation was to characterize differential right atrial (RA) and ventricular (RV) molecular changes in Ca(2+)-handling proteins consequent to RV pressure overload and hypertrophy in two common, yet distinct models of pulmonary hypertension: dehydromonocrotaline (DMCT) toxicity and pulmonary artery (PA) banding. METHODS: A total of 18 dogs underwent sternotomy in four groups: (1) DMCT toxicity (n = 5), (2) mild PA banding over 10 wk to match the RV pressure rise with DMCT (n = 5); (3) progressive PA banding to generate severe RV overload (n = 4); and (4) sternotomy only (n = 4). RESULTS: In the right ventricle, with DMCT, there was no change in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) or phospholamban (PLB), but we saw a trend toward down-regulation of phosphorylated PLB at serine-16 (p[Ser-16]PLB) (P = 0.07). Similarly, with mild PA banding, there was no change in SERCA or PLB, but p(Ser-16)PLB was down-regulated by 74% (P < 0.001). With severe PA banding, there was no change in PLB, but SERCA fell by 57% and p(Ser-16)PLB fell by 67% (P < 0.001). In the right atrium, with DMCT, there were no significant changes. With both mild and severe PA banding, p(Ser-16)PLB fell (P < 0.001), but SERCA and PLB did not change. CONCLUSIONS: Perturbations in Ca(2+)-handling proteins depend on the degree of RV pressure overload and the model used to mimic the RV effects of pulmonary hypertension. They are similar, but blunted, in the atrium compared with the ventricle.

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium.

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/µg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.

Identification of CaMKII phosphorylation sites in Connexin43 by high-resolution mass spectrometry.

Connexin43 (Cx43) is a major cardiac gap junction channel protein required for normal electrical and contractile activity. Gap junction channel assembly, function, and turnover are regulated by phosphorylation under both normal and disease conditions. The carboxyl terminus (CT) of Cx43 contains numerous amino acid residues that are phosphorylated by protein kinases. However, our knowledge of the specific residues and kinases involved is incomplete. The objective of this study was to identify amino acid residues in the Cx43-CT that are targets of the multifunctional protein kinase, Ca(2+)/calmodulin protein kinase II (CaMKII), an enzyme known to play critical roles in Ca(2+) homeostasis, transcription, apoptosis, and ischemic heart disease. We subjected fusion protein containing the Cx43-CT to phosphorylation by CaMKII in vitro, digestion with Lys-C and trypsin followed by enrichment for phosphorylated peptides using TiO(2), and analysis in an LTQ XL Orbitrap with collision-induced dissociation and electron transfer dissociation. We deduced the sites of modification by interpreting tandem spectra from these "orthogonal" methods of gas phase peptide fragmentation. We have identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts.

Remodeling of cardiac fibroblasts following myocardial infarction results in increased gap junction intercellular communication.

BACKGROUND: We have recently shown that native murine ventricular fibroblasts express both connexin43 (Cx43) and Cx45, and that the level of Cx43 expression influences intercellular coupling and cell proliferation. Relatively little is known, however, about how myocardial infarction (MI) influences expression of Cx43, or how altered Cx43 expression may affect fibroblast function post-MI. Fibroblasts are critical for infarct healing and post-infarct ventricular remodeling. They can couple electrically with cardiac myocytes and influence myocardial activation patterns. Thus, Cx43 remodeling and the level of intercellular communication in fibroblasts expressed in the infarcted heart were the subject of the present investigation. METHODS: Fibroblasts were isolated from both infarct scar and remote, noninfarcted regions of murine hearts 6 d after coronary ligation. Expression levels of Cx43, α-smooth muscle actin and N-cadherin were quantified by immunoblotting. Gap junctional intercellular communication was quantified by Lucifer yellow dye transfer. RESULTS AND CONCLUSIONS: Fibroblasts isolated from infarcted hearts exhibited marked up-regulation of Cx43 protein expression and enhanced intercellular coupling. Exogenous administration of transforming growth factor-ß (TGF-ß) to fibroblast cultures from normal, non-operated hearts produced comparable up-regulation of Cx43, suggesting that increased intercellular communication between fibroblasts in infarct and peri-infarct regions may be secondary to activation of a TGF-ß pathway. Unlike cardiac myocytes that down-regulate Cx43, presumably to limit intercellular transmission of biochemical mediators of ischemic injury, fibroblasts may up-regulate Cx43 to maintain electrical and metabolic coupling at a time when intercellular communication is compromised.

Reduced expression of Cx43 attenuates ventricular remodeling after myocardial infarction via impaired TGF-beta signaling.

In addition to mediating cell-to-cell electrical coupling, gap junctions are important in tissue repair, wound healing, and scar formation. The expression and distribution of connexin43 (Cx43), the major gap junction protein expressed in the heart, are altered substantially after myocardial infarction (MI); however, the effects of Cx43 remodeling on wound healing and the attendant ventricular dysfunction are incompletely understood. Cx43-deficient and wild-type mice were subjected to proximal ligation of the anterior descending coronary artery and followed for 6 days or 4 wk to test the hypothesis that reduced expression of Cx43 influences wound healing, fibrosis, and ventricular remodeling after MI. We quantified the progression of infarct healing by measuring neutrophil expression, collagen content, and myofibroblast expression. We found significantly reduced transformation of fibroblasts to myofibroblasts at 6 days and significantly reduced collagen deposition both in the infarct at 6 days and at 4 wk in the noninfarcted region of Cx43-deficient mice. As expected, transforming growth factor (TGF)-beta, a profibrotic cytokine, was dramatically upregulated in MI hearts, but its phosphorylated comediator (pSmad) was significantly downregulated in the nuclei of Cx43-deficient hearts post-MI, suggesting that downstream signaling of TGF-beta is diminished substantially in Cx43-deficient hearts. This diminution in profibrotic TGF-beta signaling resulted in the attenuation of adverse structural remodeling as assessed by echocardiography. These findings suggest that efforts to enhance the expression of Cx43 to maintain intercellular coupling or reduce susceptibility to arrhythmias should be met with caution until the role of Cx43 in infarct healing is fully understood.

Connexin43 expression levels influence intercellular coupling and cell proliferation of native murine cardiac fibroblasts.

Little is known about connexin expression and function in murine cardiac fibroblasts. The authors isolated native ventricular fibroblasts from adult mice and determined that although they expressed both connexin43 (Cx43) and connexin45 (Cx45), the relative abundance of Cx45 was greater than that of Cx43 in fibroblasts compared to myocytes, and the electrophoretic mobility of both Cx43 and Cx45 differed in fibroblasts and in myocytes. Increasing Cx43 expression by adenoviral infection increased intercellular coupling, whereas decreasing Cx43 expression by genetic ablation decreased coupling. Interestingly, increasing Cx43 expression reduced fibroblast proliferation, whereas decreasing Cx43 expression increased proliferation. These data demonstrate that native fibroblasts isolated from the mouse heart exhibit intercellular coupling via gap junctions containing both Cx43 and Cx45. Fibroblast proliferation is inversely related to the expression level of Cx43. Thus, connexin expression and remodeling is likely to alter fibroblast function, maintenance of the extracellular matrix, and ventricular remodeling in both normal and diseased hearts.

Role of activated CaMKII in abnormal calcium homeostasis and I(Na) remodeling after myocardial infarction: insights from mathematical modeling.

Ca(2+)/calmodulin-dependent protein kinase II is a multifunctional serine/threonine kinase with diverse cardiac roles including regulation of excitation contraction, transcription, and apoptosis. Dynamic regulation of CaMKII activity occurs in cardiac disease and is linked to specific disease phenotypes through its effects on ion channels, transporters, transcription and cell death pathways. Recent mathematical models of the cardiomyocyte have incorporated limited elements of CaMKII signaling to advance our understanding of how CaMKII regulates cardiac contractility and excitability. Given the importance of CaMKII in cardiac disease, it is imperative that computer models evolve to capture the dynamic range of CaMKII activity. In this study, using mathematical modeling combined with biochemical and imaging techniques, we test the hypothesis that CaMKII signaling in the canine infarct border zone (BZ) contributes to impaired calcium homeostasis and electrical remodeling. We report that the level of CaMKII autophosphorylation is significantly increased in the BZ region. Computer simulations using an updated mathematical model of CaMKII signaling reproduce abnormal Ca(2+) transients and action potentials characteristic of the BZ. Our simulations show that CaMKII hyperactivity contributes to abnormal Ca(2+) homeostasis and reduced action potential upstroke velocity due to effects on I(Na) gating kinetics. In conclusion, we present a new mathematical tool for studying effects of CaMKII signaling on cardiac excitability and contractility over a dynamic range of kinase activities. Our experimental and theoretical findings establish abnormal CaMKII signaling as an important component of remodeling in the canine BZ.

Protein kinase Cepsilon mediates salutary effects on electrical coupling induced by ischemic preconditioning.

BACKGROUND: Ischemic preconditioning delays the onset of electrical uncoupling and prevents loss of the primary ventricular gap junction protein connexin 43 (Cx43) from gap junctions during subsequent ischemia. OBJECTIVE: To test the hypothesis that these effects are mediated by protein kinase C epsilon (PKCepsilon), we studied isolated Langendorff-perfused hearts from mice with homozygous germline deletion of PKCepsilon (PKCepsilon-KO). METHODS: Cx43 phosphorylation and distribution were measured by quantitative immunoblotting and confocal microscopy. Changes in electrical coupling were monitored using the 4-electrode technique to measure whole-tissue resistivity. RESULTS: The amount of Cx43 located in gap junctions, measured by confocal microscopy under basal conditions, was significantly greater in PKCepsilon-KO hearts compared with wild-type, but total Cx43 content measured by immunoblotting was not different. These unanticipated results indicate that PKCepsilon regulates subcellular distribution of Cx43 under normal conditions. Preconditioning prevented loss of Cx43 from gap junctions during ischemia in wild-type but not PKCepsilon-KO hearts. Specific activation of PKCepsilon, but not PKCdelta, also prevented ischemia-induced loss of Cx43 from gap junctions. Preconditioning delayed the onset of uncoupling in wild-type but hastened uncoupling in PKCepsilon-KO hearts. Cx43 phosphorylation at the PKC site Ser368 increased 5-fold after ischemia in wild-type hearts, and surprisingly, by nearly 10-fold in PKCepsilon-KO hearts. Preconditioning prevented phosphorylation of Cx43 in gap junction plaques at Ser368 in wild-type but not PKCepsilon-KO hearts. CONCLUSION: Taken together, these results indicate that PKCepsilon plays a critical role in preconditioning to preserve Cx43 signal in gap junctions and delay electrical uncoupling during ischemia.

Diminished zonula occludens-1 expression in the failing human heart.

BACKGROUND: Reduced expression of the major gap junction protein connexin 43 (Cx43) in the failing human heart may lead to arrhythmias and sudden cardiac death. Cx43 interacts with the actin binding protein, zonula occludens-1 (ZO-1), and it has recently been demonstrated that ZO-1 regulates the formation and function of Cx43 gap junctions. We hypothesize that normal expression of ZO-1 and its interaction with Cx43 are required for appropriate assembly and function of Cx43 gap junctions in the heart. Here, we determined whether expression of ZO-1 is altered in patients with heart failure. METHODS: We examined ventricular myocardium from hearts of patients in end-stage heart failure, obtained at transplant, for ZO-1 expression by immunohistochemistry. We also subjected lysates made from this tissue to immunoblotting to determine the level of ZO-1 expression. RESULTS AND CONCLUSIONS: ZO-1 was found at 96% of the intercalated discs in nonfailing control human hearts, where it colocalized with Cx43. In contrast, there was ZO-1 immunostaining at 5% of intercalated discs in failing hearts, coincident with a reduction in Cx43 staining in intercalated discs. Immunoblotting analysis showed that there was a 95% reduction in ZO-1 expression in human heart failure. Loss of ZO-1 at intercalated discs in heart failure may play a critical role in remodeling of Cx43 gap junctions, which may contribute to abnormal impulse propagation and arrhythmogenesis, thereby predisposing patients in heart failure to sudden cardiac death.

Arrhythmia susceptibility and premature death in transgenic mice overexpressing both SUR1 and Kir6.2[DeltaN30,K185Q] in the heart.

Sarcolemmal ATP-sensitive potassium (K(ATP)) channels are activated after pathological depletion of intracellular ATP, unlike their pancreatic beta-cell counterparts, which dynamically regulate membrane excitability in response to changes in blood glucose. We recently engineered a series of transgenic (TG) mice overexpressing an ATP-insensitive inward rectifying K(+) channel protein (Kir)6.2 mutant (Kir6.2[DeltaN30,K185Q]) or the accessory sulfonylurea receptor (SUR)2A (FLAG-SUR2A) or SUR1 (FLAG-SUR1) subunits of the K(ATP) channel, under transcriptional control of the alpha-myosin heavy chain promoter. In the present study, we generated double transgenic (DTG) animals overexpressing both Kir6.2[DeltaN30,K185Q] and FLAG-SUR1 or FLAG-SUR2A and examined the effects on cardiac excitability in vivo. No animals expressing both FLAG-SUR1 and Kir6.2[DeltaN30,K185Q] transgenes at a high level were obtained. DTG mice expressing one transgene at a high level and the other at a lower level are born, but they die prematurely. Electrocardiographic analysis of both anesthetized and conscious animals revealed a constellation of arrhythmias in DTG animals, but not in wild-type or single TG littermates. The proarrhythmic effect of the transgene combination is intrinsic to the myocardium, since it persists in isolated hearts. Importantly, this effect is specific for SUR1-expressing DTG animals: DTG animals expressing both Kir6.2[DeltaN30,K185Q] and FLAG-SUR2A at high levels exhibit neither impaired survival nor increased arrhythmia frequency, even with both subunits expressed at high levels. In demonstrating the profound arrhythmic consequences of K(ATP) channels comprised of SUR1 and Kir6.2[DeltaN30,K185Q] in the myocardium specifically, the results highlight the critical differential activation of SUR1 versus SUR2A, and indicate that expression of hyperactive K(ATP) in the heart is likely to be proarrhythmic.