Background:Both Nav1.5 and Cx43 are critical for the fast electrical impulse conduction in the myocardium and their reductions create the arrhythmogenic substrate. Wnt/β-catenin signaling is activated in arrhythmogenic myocardium, and although this signaling is known to downregulate cardiac Nav1.5, its regulation of Cx43 is unclear as conflicting results have been reported. The present study investigated how Wnt/β-catenin signaling regulates Cx43 in rat and human cardiomyocytes and if it is dependent on the sex of the cells or the metabolic substrates. Methods:Male and female neonatal rat ventricular myocytes (NRVMs) were treated with CHIR-99021 (CHIR) or Wnt3a protein, two different activators of the Wnt/β-catenin signaling, either in a medium rich in glucose (a preferred metabolic substrate in heart failure) or in a medium rich in lipid (∼150 μM fatty acid, a preferred substrate in healthy hearts). Both healthy and Brugada Syndrome human iPSC-derived cardiomyocytes (iPSC-CMs) were used to confirm observations in NRVMs. Results:When maintained in a glucose-rich medium, Gja1 mRNA (encoding Cx43) was reduced by a low concentration of CHIR (1 μM) in female NRVMs but only at a high concentration of CHIR (10 μM) in male NRVMs. However, reductions in Cx43 protein were observed at 1 μM CHIR in both male and female NRVMs, suggesting the involvement of both transcriptional and post-transcriptional mechanisms. When maintained in a lipid-rich medium, neither Gja1 mRNA nor Cx43 protein was altered by CHIR at 1 or 3 μM. In contrast, CHIR-induced reductions in Scn5a mRNA and Nav1.5 protein were observed in both glucose-rich and lipid-rich media, with no significant sex-specific differences detected. Consistent with studies using CHIR, which is a Wnt receptor-independent activator, Wnt3a protein also reduced both Gja1 mRNA and Cx43 protein in NRVMs in the glucose-rich medium but not in the lipid-rich medium. In human iPSC-CMs from two healthy volunteers and one Brugada Syndrome patient, Wnt/β-catenin signaling activation reduced GJA1 mRNA and Cx43 protein in a standard, glucose-containing medium. Conclusions:These data demonstrate that metabolic substrates regulate the effects of Wnt/β-catenin signaling in cardiomyocytes, with reductions in Cx43 mRNA and protein only observed when glucose is the primary metabolic substrate, which occurs in arrhythmogenic conditions such as cardiac hypertrophy and heart failure.

Wnt/β-catenin signaling has been shown to regulate gene expressions in cardiomyocytes. However, it is not known if this effect is dependent on the sex of cells or the glucose level in the culture medium. In the present study, ventricular myocytes were prepared from male and female neonatal rats and maintained in either a glucose-rich (25 mM) medium or a low-glucose (3 mM), lipid-rich medium. Real-time quantitative PCR was used to measure changes in target genes (Axin2, Scn5a, and Tbx3) after treatment with 1, 3, or 10 µM of CHIR-99021, an activator of Wnt/β-catenin signaling. CHIR induced similar changes in Axin2, Tbx3, and Scn5a transcripts in male and female NRVMs in both media, suggesting the absence of sex difference. However, cells in a high-glucose medium showed greater increases in Axin2 and Tbx3 transcripts than cells in a low-glucose medium. In addition, a low concentration of CHIR (1 µM) reduced the Scn5a transcript in cells in a high-glucose medium but not in a low-glucose medium, suggesting an increased sensitivity to Wnt signaling by high glucose. A non-linear relationship was identified between Axin2 transcript upregulation and Scn5a transcript downregulation in CIHR-treated NRVMs. These data suggest that high glucose sensitizes both male and female cardiomyocytes to Wnt/β-catenin signaling.

Aims: Calcium currents are critical for the excitation-contraction coupling of atrial and ventricular myocytes and for the pacemaker function of the sinoatrial node (SAN). The Wnt/β-catenin signaling is activated in heart failure that is frequently associated with SAN dysfunction. However, it is unknown if Wnt/β-catenin signaling regulates calcium currents in cardiomyocytes and affects SAN function. Methods and Results: Neonatal rat ventricular myocytes (NRVMs), which express both L-type and T-type calcium currents (ICa,L and ICa,T), were treated with either Wnt3a protein or CHIR-99021 (CHIR), two different activators of the Wnt/β-catenin signaling. PCR array, qPCR, western blot and whole-cell patch-clamp recording showed that the transcript, protein, and current density of the T-type calcium channel (Cacna1g and Cav3.1), but not the L-type calcium channel (Cacna1c and Cav1.2), were reduced in NRVMs by Wnt3a or CHIR treatment. In sinoatrial node tissues isolated from adult rats and mice, the spontaneous beating rates were reduced by ML-218, a blocker of ICa,T, suggesting a role of this current in sinoatrial node pacemaking. A gel containing CHIR was applied to the sinoatrial node of adult rats to activate Wnt/β-catenin signaling. The spontaneous beating rate of sinoatrial nodes was lower with frequent pauses in CHIR-painted rats as compared to DMSO-painted control rats, suggesting sinoatrial node dysfunction after Wnt/β-catenin signaling activation. In addition, both Cav3.1 protein and T-type calcium current were reduced in single sinoatrial node pacemaker myocytes isolated from CHIR-painted rats as compared to cells from DMSO-painted control rats. Conclusions: This study demonstrated for the first time that Wnt/β-catenin signaling inhibits T-type calcium current in cardiomyocytes and causes sinoatrial node dysfunction.

Abstract The voltage‐gated Nav1.5 channels mediate the fast Na+ current (INa) in cardiomyocytes initiating action potentials and cardiac contraction. Downregulation of INa, as occurs in Brugada syndrome (BrS), causes ventricular arrhythmias. The present study investigated whether the Wnt/β‐catenin signaling regulates Nav1.5 in human‐induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs). In healthy male and female iPSC‐CMs, activation of Wnt/β‐catenin signaling by CHIR‐99021 reduced (p < 0.01) both Nav1.5 protein and SCN5A mRNA. In iPSC‐CMs from a BrS patient, both Nav1.5 protein and peak INa were reduced compared to those in healthy iPSC‐CMs. Treatment of BrS iPSC‐CMs with Wnt‐C59, a small‐molecule Wnt inhibitor, led to a 2.1‐fold increase in Nav1.5 protein (p = 0.0005) but surprisingly did not affect SCN5A mRNA (p = 0.146). Similarly, inhibition of Wnt signaling using shRNA‐mediated β‐catenin knockdown in BrS iPSC‐CMs led to a 4.0‐fold increase in Nav1.5, which was associated with a 4.9‐fold increase in peak INa but only a 2.1‐fold increase in SCN5A mRNA. The upregulation of Nav1.5 by β‐catenin knockdown was verified in iPSC‐CMs from a second BrS patient. This study demonstrated that Wnt/β‐catenin signaling inhibits Nav1.5 expression in both male and female human iPSC‐CMs, and inhibition of Wnt/β‐catenin signaling upregulates Nav1.5 in BrS iPSC‐CMs through both transcriptional and posttranscriptional mechanisms.

Background: The T-type Ca 2+ channels (I Ca,T ) are expressed in the fetal and neonatal cardiomyocytes, but in the adult heart they are exclusively expressed in the cardiac conduction system, including the sinoatrial node (SAN). In contrast, the L-type Ca 2+ channels (I Ca,L ) are expressed ubiquitously in the adult heart and are important for cardiac contraction. The Wnt/β-catenin signaling (Wnt signaling) is activated in various heart disease and we and others have recently demonstrated that Wnt signaling inhibits cardiac Na + current. In the present study, we investigated if calcium channels are also regulated by Wnt signaling. Methods and Results: Because neonatal rat ventricular myocytes (NRVMs) express both I Ca,L and I Ca,T , we investigated the effect of Wnt signaling on NRVMs by treatment with either Wnt3a protein or CHIR-99021 (CHIR) (two different activators of the Wnt signaling). PCR array and RNA sequencing studies showed that Wnt signaling did not affect the mRNA of Cacna1c (encoding the α subunit of I Ca,L , Ca v 1.2), but the mRNA of Cacna1g (encoding the α subunit of I Ca,T , Ca v 3.1) was reduced in NRVMs (p<0.05, n=3) after treatment with either Wnt3a or CHIR. These observations were verified by qRT-PCR which showed dose-dependent reductions (p<0.05, n=4) in Cacna1g mRNA after CHIR treatment. Western blot and patch-clamp also demonstrated reductions (p<0.01, n=5) in channel protein and ionic current density of T-type, but not L-type, Ca 2+ channels after treatment with Wnt3a or CHIR. These observations suggest that Wnt signaling selectively inhibits T-type Ca 2+ channels in cardiomyocytes. Consistent with a critical role of T-type Ca 2+ channels in the pacemaker activity of SAN (the primary pacemaker of the heart), the spontaneous firing rates of isolated rat and mouse SAN tissues were reduced by ML-218 (an I Ca,T blocker) in a dose-dependent manner (p<0.01, n=5). In addition, activation of Wnt signaling in adult rat SAN tissues by CHIR led to slower and irregular beating, indicative of SAN dysfunction (p<0.01, n=5). Conclusions: These observations are consistent with the conclusions that Wnt/β-catenin signaling selectively inhibits T-type calcium current and may play a role in sinoatrial node dysfunction.

Heart disease is the leading cause of morbidity and mortality worldwide. Due to their low cost, ease of handling, and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

BACKGROUND: Survivors of community-acquired pneumonia (CAP) are at increased risk of cardiovascular disease, cognitive and functional decline, and death, but the mechanisms remain unknown. RESEARCH QUESTION: Do CAP survivors have evidence of increased inflammatory activity in their lung parenchyma on 2-deoxy-2-[18F]fluoro-D-glucose (18FDG)-PET/CT imaging after clinical resolution of infection? STUDY DESIGN AND METHODS: We obtained 18FDG-PET/CT scans from 22 CAP survivors during their hospitalization with pneumonia (acute CAP) and 30 to 45 days after hospital discharge (post-CAP). In each set of scans, we assessed the lungs for foci of increased 18FDG uptake by visual interpretation and by total pulmonary glycolytic activity (tPGA), a background-corrected measure of total metabolic activity (as measured by 18FDG uptake). We also measured, post-CAP, the glycolytic activity of CAP survivor lung areas with volumes similar to the areas in 28 matched historical control subjects without pneumonia. RESULTS: Overall, 68% of CAP survivors (95% CI, 45%-85%) had distinct residual areas of increased 18FDG uptake in their post-CAP studies. tPGA decreased from 821.5 (SD, 1,140.2) in the acute CAP period to 80.0 (SD, 81.4) in the post-CAP period (P 1/4 .006). The tPGA post-CAP was significantly higher than that in lung areas of similar volume in control subjects (80.0 [SD, 81.4] vs -19.4 [SD, 5.9]; P < .001). INTERPRETATION: An important proportion of CAP survivors have persistent pulmonary foci of increased inflammatory activity beyond resolution of their infection. As inflammation contributes to cardiovascular disease, cognitive decline, functional waning, and mortality risk in the general population, this finding provides a plausible mechanism for the increased morbidity and mortality that have been observed post-CAP. CHEST 2021; 160(2):446-453

Wnt/β-catenin signaling is activated in the heart after myocardial infarction (MI). This study aims to investigate if β-catenin deletion affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at 8 weeks after MI, associated with reduced scar size and attenuated chamber dilation. qPCR analyses of both myocardial tissues and purified cardiomyocytes demonstrated upregulation of Wnt pathway genes in border and infarct regions after MI, including Wnt ligands (such as Wnt4 ) and receptors (such as Fzd1 and Fzd2 ). At 1 week after MI, cardiac sodium channel gene ( Scn5a) transcript was reduced in WT but not in KO hearts, consistent with previous studies showing Scn5a inhibition by Wnt/β-catenin signaling. At 8 weeks after MI when Wnt genes have declined, Scn5a returned to near sham levels and K + channel gene downregulations were not different between WT and KO mice. This study demonstrated that VT susceptibility in the chronic phase after MI is reduced in mice with cardiomyocyte-specific β-catenin deletion primarily through attenuated structural remodeling, but not ion channel gene alterations.

Background: Wnt signaling plays a critical role in both embryonic cardiogenesis and cardiac remodeling in adult heart disease. We have previously demonstrated that the canonical Wnt/β-catenin pathway inhibits cardiac sodium current, but it remains unclear whether the noncanonical Wnt pathway affects cardiac electrophysiology. Methods and Results: Western blot analysis of ventricular tissues from patients with heart failure (n=6) demonstrated a 2.3x fold increase (p<0.01) in the protein level of Wnt5a, a noncanonical Wnt ligand, as compared to healthy ventricular tissues (n=5). To investigate if Wnt5a affects cardiac electrophysiology, adenovirus expressing Wnt5a and mCherry (Ad-Wnt5a) or control adenovirus expressing mCherry only (Ad-mCherry) was injected into the left ventricular free wall of adult rat hearts. At 4-5 days after virus injection, surface ECG revealed increased QT interval (p<0.01) in Ad-Wn5a-injected rats (90.1±2.3 ms n=7, vs 72.3±2.0 ms in control Ad-mCherry rats n=7). In addition, ventricular tachycardia was induced by programmed electrical stimulation in 92% (11/12) Ad-Wnt5a hearts, but only in 22% (2/9) control Ad-mCherry hearts (p<0.01). Patch-clamp recording of isolated single ventricular myocytes demonstrated that Ad-Wnt5a myocytes exhibited marked prolongation of action potential duration (APD 90 : 273±77ms, n=5) as compared to control cells (42±12 ms, n=7, p<0.05). In addition, the prolonged action potentials in Ad-Wnt5a myocytes were associated with frequent early afterdepolarizations and delayed afterdepolarizations, two mechanisms for triggered ventricular arrhythmias. Conclusion: Wnt5a is increased in the myocardium of patients with heart failure. Viral expression of Wnt5a in rat ventricular tissue increases QT interval and ventricular arrhythmia susceptibility, which is associated with prolongation of action potentials in cardiomyocytes. This may be an important target for future therapies.

The voltage-gated Nav1.5 channels mediate the fast Na+ current (INa) in cardiomyocytes initiating action potentials and cardiac contraction. Downregulation of INa, as occurs in Brugada Syndrome (BrS), causes ventricular arrhythmias. The present study aims to investigate whether the Wnt/-catenin signalling (Wnt signalling) regulates Nav1.5 expression in human cardiomyocytes and whether inhibition of Wnt signalling upregulates Nav1.5 in BrS cardiomyocytes. In human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that were derived from two healthy volunteers (one male and one female), pharmacological activation of Wnt/-catenin signalling via CHIR-99021 treatment reduced (p<0.01) both Nav1.5 protein and SCN5A mRNA. In iPSC-CMs derived from a BrS patient, both Nav1.5 protein and peak INa density were reduced compared to those in healthy iPSC-CMs. Treatment of BrS iPSC-CMs with Wnt-C59, a small-molecule inhibitor of Wnt signalling, led to a 2.1-fold increase in Nav1.5 protein (p=0.0005) but surprisingly did not affect SCN5A mRNA (p=0.146). Markers of myocyte maturation, α-sarcomeric actinin protein, Kir2.1 protein and KCNJ2 mRNA were not affected by Wnt-C59. Similarly, inhibition of Wnt signalling using shRNA-mediated -catenin knockdown in BrS iPSC-CMs led to a 4.4-fold increase in Nav1.5, which was associated with a 4.9-fold increase in peak INa density but only a 2.1-fold increase in SCN5A mRNA. The upregulation of Nav1.5 by -catenin knockdown was verified in iPSC-CMs derived from a second BrS patient. This study demonstrated that Wnt/-catenin signalling inhibits Nav1.5 expression in both male and female human iPSC-CMs, and inhibition of Wnt signalling upregulates Nav1.5 in BrS iPSC-CMs through both transcriptional and post-transcriptional mechanisms.

Introduction: Cardiovascular disease (CVD) is a major source of morbidity and mortality in HIV+ patients, which is thought to be in part due to a state of persistent systemic inflammation that results in accelerated atherosclerosis. Hypothesis: We hypothesized that inflammation, as measured by 18F-fluorodeoxyglucose (18F-FDG) uptake measured by 18F-FDG positron emission tomography/computed tomography (18F-FDG-PET/CT) in the bone marrow, spleen and thoracic aorta would improve in HIV+ patients following therapy with rosuvastatin compared to HIV+ patients not receiving a statin. Methods: HIV+ patients with a moderate Framingham risk score were enrolled in the study and randomized to treatment with rosuvastatin or usual treatment for 6 months. Patients were matched for age, sex, smoking status, Framingham risk score, duration of anti-retroviral therapy (ART) and type of ART. Fasting blood was collected and 18F-FDG-PET/CT imaging of bone marrow, spleen, and thoracic aorta was performed at baseline and 6 months. Results: Thirty-five HIV+ patients were enrolled in the study; 17 were randomized to treatment with rosuvastatin and 18 were randomized to the control group. There was a significant drop in the 18F-FDG bone marrow, spleen and thoracic aortic uptake in the statin-treated group compared to the control group (bone marrow: -10.3±16.9% versus 5.0±18.9%, p=0.0262; spleen: -9.8±20.3% versus 11.3±28.8%, p=0.0497; thoracic aorta: -8.6±24.5% versus 12.6±29.5%, p=0.0343). 18F-FDG changes over the study period are shown in Figure 1. Conclusions: The present study observed a significant decrease in 18F-FDG-PET uptake in the bone marrow, spleen, and thoracic aorta following treatment with rosuvastatin for 6 months in HIV+ patients. These data suggest a wide-range anti-inflammatory effect of rosuvastatin in HIV+ individuals with well-controlled infection on ART, ultimately resulting in decreased inflammatory activity in the arterial walls of these patients.

The Wnt/β-catenin signaling regulates ion channel gene expressions in cardiomyocytes. Because Wnt/β-catenin signaling is activated in myocardial infarction (MI), this study aims to investigate if β-catenin inhibition affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at week-8 after MI, associated with attenuated structural remodeling (reduced scar size and attenuated left ventricle dilation) as compared to WT. However, at the subacute phase (week-1) and chronic phase (week-8) after MI, Wnt/β-catenin signaling activation was found in non-cardiomyocytes, but not in cardiomyocytes. Downregulations of Scn5a (encoding Na v 1.5) and Gja1 (encoding Cx43) were found at week-1 but not at week-8, while downregulations of K + channel genes were present at both week-1 and -8. Consistent with no activation of Wnt/β-catenin pathway in cardiomyocytes at week-1 and -8, these alterations in ion channel/transporter genes were not different between KO and WT mice. This study demonstrated that mice with cardiomyocyte-specific β-catenin deletion have reduced VT susceptibility after MI which is caused by attenuated structural remodeling, instead of alterations in ion channel gene expressions.

BACKGROUND:Myocardial infarction and heart failure are associated with reduced voltage-gated Na+ current (INa) that promotes arrhythmias and sudden deaths. We have previously shown that the Wnt/β-catenin signalling (Wnt signalling), which is active in heart disease, reduces cardiac INa, suggesting that Wnt signalling may be a potential therapeutic target. However, because Wnt signalling is required for the homeostasis of many noncardiac tissues, administration of Wnt inhibitors to heart patients would cause significant side effects. The present study aims to elucidate the molecular mechanisms of cardiac INa inhibition by Wnt, which would identify cardiac-specific therapeutic targets.METHODS:Wnt signalling was activated in neonatal rat ventricular myocytes by Wnt3a protein. Adenovirus expressing Wnt3a was injected into the adult rat ventricle. CRISPR/Cas9 and chromatin immunoprecipitation were used for mechanistic studies.RESULTS:Wnt signalling activation in neonatal rat ventricular myocytes reduced Nav1.5 protein and Scn5a mRNA, but increased Tbx3, a known suppressor of Scn5a. Chromatin immunoprecipitation showed that Wnt signalling inhibits Scn5a expression through downstream mediator (TCF4) binding to both Tbx3 and Scn5a promoters. Overexpression or knockdown of Tbx3 directly modified Nav1.5 and INa, whereas CRISPR/Cas9-induced mutations at TCF4 binding sites within the Scn5a promoter attenuated Wnt inhibition of Scn5a and Nav1.5. In adult rat hearts, adenovirus expressing Wnt3a reduced Nav1.5, increased QRS duration in electrocardiogram, and increased the susceptibility to ventricular tachycardia.CONCLUSIONS:Wnt signalling inhibits the Na+ channel by direct and indirect (via Tbx3) suppression of Scn5a transcription. Strategies to block TCF4 binding to the Tbx3 and Scn5a promoters would represent novel strategies for cardiac-specific inhibition of the Wnt pathway to rescue INa and prevent sudden cardiac deaths.

Corrales-Medina, Vicente F.; Habibi, Charlene M.; Wang, Jerry; Glassman, Steven J.; MacPherson, Paul; Beanlands, Rob SB; deKemp, Rob A.; Dwivedi, Girish Author Information

Aims: Both inherited arrhythmogenic diseases (such as Brugada Syndrome) and heart failure are associated with reduced voltage-gated Na + current (I Na ) which promotes lethal arrhythmias and sudden deaths. We and others have shown that Wnt/β-catenin signaling (Wnt signaling), which is active in heart disease, inhibits I Na in rat and mouse cardiomyocytes. But whether Wnt signaling regulates I Na in human cardiomyocytes and represents a novel therapeutic target is unknown. This study aims to investigate if Wnt signaling inhibits I Na in human cardiomyocytes, to elucidate the underlying mechanisms, and to test if blocking Wnt signaling can rescue I Na in Brugada Syndrome patient cardiomyocytes. Methods and Results: Cardiomyocytes were differentiated from human induced pluripotent stem cells (hiPSC-CMs) that were derived from healthy volunteers or a Brugada Syndrome patient. Whole-cell patch-clamp technique was used for I Na measurement. Activation of Wnt signaling in healthy hiPSC-CMs led to a 69% reduction in I Na amplitude (peak current at -20 mV: -10.5±2.8 pA/pF, n=9 cells vs. control -33.6±4.1 pA/pF, n=6 cells, p<0.01) by reducing SCN5A mRNA (encoding the pore-forming α subunit of I Na , Na v 1.5). In addition, Wnt signaling also reduced Na v 1.5 glycosylation causing a depolarizing shift of I Na activation curve (a mechanism found in human, but not in rodent, cardiomyocytes). Blocking Wnt signaling in Brugada hiPSC-CMs with shRNA-mediated β-catenin knockdown led to a 13-fold increase in I Na amplitude (p<0.01, n=9 cells), offsetting the fundamental genetic defect. Consistent with increased I Na, blocking Wnt signaling also upregulated SCN5A mRNA and Na v 1.5 protein, without affecting expression of other cardiac ion channels in Brugada hiPSC-CMs. Conclusions: This study demonstrated Wnt-inhibition of human cardiac I Na and, using Brugada Syndrome as an example, demonstrated that blocking Wnt signaling is a novel therapeutic strategy to rescue I Na in heart disease.