B-Raf is required for normal murine cardiac development and function.

Objectives: The postnatal heart does not retain the proliferative capacity it had during fetal life. Unlike large mammals, murine cardiomyocytes (CM) continue to divide into the first week of life before terminal differentiation and binucleation. We hypothesized that B-Raf regulates ERK activation in newborn CM and that loss of B-Raf suppresses cyclin levels and reduced proliferation. To test this, we determined whether loss of B-Raf disrupts the ERK (extracellular-regulated kinase) cascade and impairs CM growth. Methods: CM specific knockout (KO) of B-Raf was generated using CRE/lox (floxed B-Raf x αMHC CRE) resulting in a truncated, unstable B-Raf and a null phenotype. KO mice (α-MHC-CRE / B-Raf lox/lox ) were compared to CRE negative / B-Raf lox/lox mice (wild type; WT). Hearts from 3d and 8d old pups were harvested for molecular analysis of B-Raf signaling and cell cycle markers. Hearts from 3d old pups were harvested and CMs isolated for culture using a trypsin/DNAse digestion. The cells were treated with Isoproterenol (Iso;10uM), forskolin (20uM), and IGF-1 (1ng/ml) for 15 min to determine if the loss of B-Raf results in reduced activation of ERK. Results: Heart weight to body weight (HW/BW) ratio was less in 3d KO versus 3d WT (n=50, p<0.05). HW/BW ratio became greater in 8d KO; there was no difference in 3d and 8d HW/BW in WT animals. Baseline B-Raf and phosphorylated ERK levels were reduced in KO hearts (*p<0.05). Cell cycle inhibitors p21 and p53 were increased in 3d KO hearts with decreased levels of all cyclins (p<0.05). In 8d KO hearts, increased p21, p27, and p53 expression was accompanied with increased cyclin levels (p<0.05). In vitro ERK activation was blunted in KO CMs by forskolin and Iso compared to IGF-1. Conclusions: ERK activation was suppressed in KO hearts resulting in smaller newborn hearts but which exceeded normal HW/BW by 8d. This is may represent premature hypertrophy as the proliferative period of CM development had ended. Cell cycle analysis supports reduced CM mitotis among 8d CM. Such early disturbances in normal CM growth may increase susceptibility for reduced cardiac function in the face of increased postnatal load stress.

Introduction: Under cardiac stress, the mammalian heart undergoes ventricular remodeling to maintain cardiac function. Although these remodeling changes are viewed as beneficial at first, they eventually lead to pathological fibrosis and apoptosis. The role of the small GTPase Septin-4 (Sept4) in regulating apoptosis and regeneration through survival of stem cells has been established in several organs. However, the role of Sept4 in the heart, an organ without stem cells, and a potential role in regulating the cardiac stress response is currently unknown. Hypothesis: Sept4 induces fibrosis and apoptosis in the mammalian heart after injury. Methods: 10-week-old Sept4 knockout (KO) and wild type (WT) mice underwent transverse aortic constriction injury (TAC). Functional and molecular analyses on KO and WT hearts were done either at baseline, 1- or 4-weeks post-injury timepoints depending on the assay. Results: Cardiac function was comparable between KO and WT mice under homeostatic conditions. After TAC injury, ejection fraction and fractional shortening were decreased significantly in WT mice but maintained in KO mice. The level of cardiomyocyte apoptosis was lower in KO hearts after injury with reduced development of fibrosis compared with WT hearts. We performed transcriptomic and proteomic analyses showing that KO hearts exhibited reduced levels of ECM deposition under both baseline and post-injury conditions. Furthermore, KO hearts were more compliant and demonstrated enhanced diastolic function compared to WT hearts. Mechanistically, Sept4 deletion blunted TGF-B signaling and Postn expression in cardiac fibroblasts after injury. In cultured cardiac fibroblasts, both Sept4 knockout and knockdown significantly decreased fibroblast activation after TGF-B treatment. Conclusions: We identified Sept4 as a crucial regulator of ECM remodeling in the heart. Sept4 deletion leads to reduced levels of cardiac fibrosis and alleviation of pressure overload-induced cardiac dysfunction. Overall, our study highlights Sept4 as a potential target to prevent pathological fibrosis in the heart.

ObjectivesThe postnatal heart does not retain the proliferative capacity it had during fetal life. Unlike large mammals, murine cardiomyocytes (CM) continue to divide into the first week of life before terminal differentiation and binucleation. This postnatal proliferation and maturation window offers the ability to study the mechanisms of CM terminal differentiation. We hypothesize that B‐Raf regulates ERK (extracellular‐regulated kinase) activation in newborn CM and that loss of B‐Raf suppresses cyclin levels and reduces CM proliferation. To test this, we determined whether the loss of B‐Raf disrupts the ERK cascade and impairs CM growth.MethodsCM specific knockout (KO) of B‐Raf was generated using CRE/lox (floxed B‐Raf x αMHC CRE) resulting in a truncated, unstable B‐Raf and a null phenotype. KO mice (α‐MHC‐CRE/B‐Raf lox/lox) were compared to CRE negative/B‐Raf lox/lox mice (wild type; WT). Hearts from 1d, 3d, 8d and 14d old pups were harvested for molecular analysis of B‐Raf signaling and cell cycle markers (p21, p27, p53, cyclins A1, B1, D1 and E). Mid‐wall sections of the heart were stained with Masson's trichrome to determine changes in tissue composition.ResultsHeart weight to body weight (HW/BW) ratio was less in 3d KO versus 3d WT (n=50, p<0.05). HW/BW ratio became greater in 8d KO; there was no difference in 3d and 8d HW/BW in WT animals. B‐Raf and phosphorylated ERK levels were reduced in KO hearts (*p<0.05). Cell cycle inhibitors p21 and p53 were increased in 3d KO hearts with decreased levels of all cyclins (Cyclin A1 and B1, p<0.05). In 8d KO hearts, increased p21, p27, and p53 expression was accompanied with decreased cyclin levels (p<0.05). In 14d KO hearts, gene expression of p21, p27, and p53 are increased (p<0.05), along with cyclins D1 and E. Tissue composition of cardiomyocytes decreased and collagen increased in 3d and 8d hearts (p<0.05).ConclusionsERK activation was suppressed in KO hearts resulting in smaller newborn hearts but which exceeded normal HW/BW by 8d. This is may represent premature hypertrophy as the proliferative period of CM development had ended. Cell cycle analysis supports reduced CM mitosis among 8d CM. Trichrome analysis supports proliferation has ended prematurely by the reduction of the CM fraction. Such early disturbances in normal CM growth may increase susceptibility for reduced cardiac function in the face of increased postnatal load stress.

Background: Cardiac Progenitor Cells (CPC) are pivotally involved in cardiac repair. Fibronectin (FN), an extracellular matrix protein, is highly expressed during cardiac development. In adult heart FN is a component of the cardiac stem cell niche and re-appears after myocardial infarction (MI). The role of FN signaling in CPC function and cardiac remodeling following MI has not been elucidated. We demonstrate here proliferative and protective effects of FN signaling in CPC as mediated by β1 integrin receptor and cardioprotective serine/threonine kinase Pim1. Methods: Cell death and proliferation of CPCs was measured using propidium iodide and CyQuant assays. Signaling pathways were analyzed by immunoblotting, qRT-PCR and siRNA depletion of targets. FN was localized in heart sections by immunohistochemistry and cardiac function assessed by echocardiography in control and conditional FN knockout (KO) mouse hearts following MI. Results: FN inhibits starvation and staurosporine induced cell death in CPCs and promotes proliferation in conjunction with induction of Pim1 expression. Protective and pro-proliferative effects of FN are abrogated by inhibition of Pim1 or deletion of β1 integrin receptor. In vivo CPC expansion correlates with FN expression following MI, and CPC localize to regions of up-regulated FN protein in the infarct. Cardiac function in control and KO mice remains equivalent up to 2 weeks post MI, however by 4 weeks KO heart function worsens compared to control as evidenced by ejection fraction values measuring 16.4 +/- 1.5% vs 26.6 +/- 1.8% at 12 weeks post MI in KO and control hearts, respectively. Conclusion: FN provides pro-survival and pro-proliferative effects to CPCs in a Pim1 kinase and β1 integrin dependent manner. CPCs and FN colocalize in vivo in an infarction injury model, while conditional KO of FN in mice leads to further impairment of cardiac function after MI. Taken together these results indicate previously unidentified cardioprotective and regenerative roles for FN in pathologically challenged heart.

Rongxue Wu, Kusum Chawla, Yoshihiko Ichikawa, Mohsen Granefar Hossein Ardehali Background: Aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of basic helix-loop-helix Per/ARNT/Sim (bHLH-PAS) proteins and serves as a binding partner for a number of other family members. ARNT is also a required dimerization partner of HIF1a. Although HIF1a is known to be required for normal cardiac development, the role of ARNT in basal cardiac function in the adult heart is not known. We hypothesized that ARNT is required for normal cardiac physiology, and its deletion in adult heart results in cardiomyopathy. Methods and Results: The deletion of ARNT gene in the heart of 3 month old mice was achieved by crossing ARNT flox/flox mice with αMHC-MCM (tamoxifen-inducible heart specific Cre) transgenic mice followed by administration of tamoxifen chow. MHC-MCM/ARNT +/+ (WT) littermates were used as a control. The ARNT knock out (KO) mice exhibited enlarged left ventricle with a significant reduction in ejection fraction (KO vs. WT: 35.8 ± 3.6% vs. 61.2 ± 2.8%, n=12, p<0.01) and fractional shortening (KO vs. WT: 18.3 ± 1.5 % vs. 30.1 ± 1.1%, p<0.01), assessed by Echocardiography. Closed-chest catheterization also demonstrated reduced +dP/dt (KO vs. WT: 4355 ± 538 vs. 9426 ± 180 mmHg, p<0.01) and increased left ventricular end diastolic pressure (KO vs. WT: 3.12 ± 1.1 vs. 8.47± 1.2 mmHg, P<0.05) in KO mice but not in WT mice. The worsened cardiac function in the KO mouse heart was associated with increase in ANF and BNP expression, interstitial fibrosis, and apoptosis as determined by TUNEL staining. Furthermore, electron microscopy reveals a variety of degenerative changes and some lipid droplet in the KO hearts, and histological studies demonstrated intramyocardial lipid accumulation in the perivascular area in the KO mouse hearts. Analysis of gene expression in the KO heart revealed up-regulation of peroxisome proliferator-activated receptor alpha. Conclusion: ARNT is essential for the maintenance of structural and functional homeostasis in the adult heart, and its inactivation leads to cardiac contractile dysfunction. Our findings implicate a novel critical transcriptional requirement for ARNT in the maintenance of adult cardiac function.

The role of miR-451 in certain cardiac diseases was recently reported including ischemia/reperfusion and hypertrophic cardiomyopathy. Oxidative stress is known to involve in the above diseases. Currently the relationship between miR-451 and cardiac oxidative stress is unknown. We thus hypothesize that miR-451 act as a key mediator for cardiac hypertrophy via regulating myocardial ROS level and antioxidant pathways. We first compared the heart/body weight (HW/BW) ratio between age- & gender-matched miR-451 knockout (KO) and wild-type (WT) mice. Increase of HW/BW was found in KO mice (6.1±0.1 vs 4.8±0.1 mg/g in WT, p<0.01). Expression of fetal genes ANF (2.9-fold↑) and β-MHC (4.0-fold↑) was also significantly increased in KO hearts, as well as the ROS level (2.3-fold↑, p<0.05) in KO hearts. Consistently, both the protein expression and activation levels of the oxidation-regulating gene Nrf2 decreased the same extent (~33%↓, p<0.05) in KO hearts. In contrast, the protein expression of the direct antioxidants HO-1 and SOD3 were found significantly increased in KO hearts, suggesting a compensatory mechanism to quench the increased cardiac ROS. Since we previously showed that phosphorylation (p) of AMPKα was inhibited in cardiac hypertrophy, thus the p-AMPKα level was tested and found significantly (76%↓, p<0.05) decreased in KO heart, consistent with the hypertrophy in KO heart. In order to further explore the relationship between Nrf2 and AMPKα, cultured adult rat ventricular myocytes (ARVM) was treated with an Nrf2 activator sulforaphane, which induced 1.4-fold increase in p-AMPKα (p<0.01); whereas the AMPK activator AICAR didn’t affect Nrf2 expression in ARVM, suggesting Nrf2 is upstream from AMPK. When miR-451 mimics were overexpressed in cultured cardiac myocytes, Nrf2 was significantly increased (2.3-fold↑, p<0.05) with overexpression of miR-451 mimics vs control, suggesting miR-451 regulates Nrf2 expression. Taken together, our data show that miR-451 activates cardiac Nrf2 and its downstream AMPK signaling pathway; whereas antioxidants HO-1 and SOD3 act as compensatory mechanism for increased cardiac ROS when miR-451 is defect. miR-451 plays a novel protective role against cardiac oxidative stress, and also mediates cardiac hypertrophy.

Chronic pressure-overload (PO) induced-dilated cardiomyopathy (DCM) is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of glycogen synthase kinase-3α (GSK-3α) in PO-induced remodeling is not clear and existing dataset with global transgenic and knockout (KO) models show opposing roles. We sought to identify the specific role of GSK-3α in PO-induced dilatative cardiac remodeling. To better understand the role of GSK-3α, we employed cardiomyocyte-specific GSK3A ( GSK3A fl/fl MerCreMer ) KO mice. Post-tamoxifen treatment, the GSK-3α KO and littermate control mice underwent sham or trans-aortic constriction (TAC) surgery. Heart function was assessed at 0, 2, 4 and 6 week post-TAC using serial M-mode echocardiography. Cardiac function in the KOs and littermate controls declines equally up to 2 weeks of TAC. At 4 week, KO hearts underwent further hypertrophy, retaining concentric LV remodeling and preserved contractile function both at systole and diastole. In contrast, wild-type LV showed significant chamber dilatation with an impaired contractility. Significantly reduced LV chamber dilatation [LVIDd(mm); 5.4±0.4 vs. 4.9±0.4, P =0.01] and preserved contractile function [LVEF(%); 22.2±12.6 vs. 40.0±18.7, P =0.02] remains same in the KO mice until the end of the study (6 wk). Furthermore, LV posterior wall thickness in the KO hearts, both at systole and diastole, were significantly greater in comparison to the controls. Consistent with preserved LV dimension, significantly less mortality was observed in the KO vs. control group during the remodeling phase. Histological analysis of heart sections further revealed better preserved LV chamber and protection against TAC-induced cellular hypertrophy in the GSK-3α KOs. Moreover, KO hearts showed significantly less fibrosis accompanied with low level of cardiomyocyte apoptosis post-6 wk of TAC. Taken together, these observations show that cardiomyocyte-specific deletion of GSK-3α protects against chronic PO-induced adverse LV remodeling and preserves contractile function. Inhibiting specifically GSK-3α using isoform-specific inhibitor could be a viable therapeutic strategy to limit the PO-induced DCM, adverse remodeling and heart failure.

Rationale: Cartilage intermediate layer protein 1 (Cilp1) is a secreted extracellular matrix (ECM) protein normally associated with bone and cartilage development. Its function and mechanism of action in adult heart disease remain elusive. Objective: To establish the function and mechanism of action of Cilp1 in post-myocardial infarction (MI) cardiac remodeling. Methods and Results: We investigated the expression of Cilp1 in mouse models of pathological cardiac remodeling and human heart failure patients. Cilp1 was expressed predominantly in cardiac fibroblasts and upregulated in response to cardiac injury and in the heart and blood of heart failure patients. We generated Cilp1 knock out (KO) and transgenic (Tg) mice with N-terminal half of the protein (NCilp1) overexpressed in myofibroblasts. Cilp1 KO mice had better cardiac function, reduced number of immune cells and myofibroblasts, and enhanced microvascular survival after MI compared to wild-type (WT) littermates. Conversely, NCilp1-Tg mice had augmented loss of cardiac function, increased number of myofibroblasts and infarct size after the MI injury. RNA-seq and gene ontology analysis indicated that cell proliferation and mTORC1 signaling were downregulated in KO hearts compared to WT hearts. In vivo BrdU labeling and immunofluorescence staining showed that myofibroblast proliferation in the Cilp1 KO heart was downregulated. Biaxial mechanical testing and ECM gene expression analysis indicated that while MI caused significant stiffness in WT hearts it had little effect on KO hearts. Upregulation of collagen expression after MI injury was attenuated in KO hearts. Recombinant CILP1 protein or NCilp1-conditioned medium promoted proliferation of neonatal rat ventricular cardiac fibroblasts via the mTORC1 signaling pathway. Conclusions: Our studies established a pathological role of Cilp1 in promoting post-MI remodeling, identified a novel function of Cilp1 in promoting myofibroblast proliferation, and suggested that Cilp1 may serve as a potential biomarker for pathological cardiac remodeling and target for fibrotic heart disease.

Background: In response to cardiac injury, the heart undergoes a remodeling process, which may be defined by a complex set of genomic, expression, molecular, cellular and interstitial changes, which can become maladaptive and contribute to the development of heart failure (HF). One of the most important components in cardiac remodeling is the development of fibrosis, which is characterized by excessive deposition of extracellular matrix (ECM) proteins by cardiac fibroblasts, which disrupts the myocardial architecture and function, thus predisposing the progression of cardiac diseases to HF. The small GTPase Septin4 (Sept4) has been described to regulate regeneration and apoptosis in several organs. However, the role of Sept4 in regulating the cardiac stress response is unknown. The present study was designed to investigate if Sept4 would be mediating the alterations associated with cardiac remodeling induced by cardiac pressure overload. Hypothesis: We hypothesized that Sept4 deletion prevents the fibrotic response associated with cardiac remodeling, and the development and progression of HF triggered by transverse aortic constriction (TAC). Methods: 10-week-old wild type (WT) and Sept4 knockout (KO) mice were subjected to TAC to induce cardiac pressure overload. Functional and molecular analyses on WT and KO hearts were performed either at baseline, 1- or 4-weeks post-injury timepoints. Results: After TAC injury, WT mice showed a significant reduction of cardiac function and the development of heart failure, while KO mice were able to maintain normal cardiac function. KO hearts exhibited decreased levels of cardiac ECM deposition and fibrosis compared with WT hearts. Furthermore, KO hearts were more compliant and demonstrated improved end diastolic pressure compared with their WT counterparts. The differentiation of fibroblasts into myofibroblasts was impaired in KO hearts compared with WT controls, which appeared to be associated with attenuated TGF-β-triggered signaling pathway in KO hearts after TAC injury. In line with these findings, we verified in cultured cardiac fibroblasts (CFBs) that Sept4 deletion resulted in reduced myofibroblasts differentiation and a blunted ability of CFBs to contract. Conclusion: Our results demonstrate that Sept4 is an important regulator of ECM remodeling in the heart. Sept4 deletion leads to reduced levels of cardiac fibrosis and attenuation of pressure overload-induced cardiac dysfunction. These findings highlight Sept4 as a potential target to prevent fibrosis in cardiac stress response. This study was supported by grants from the National Institutes of Health (HL155993 and HL160665). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Molecular Alterations in a Mouse Cardiac Model of Friedreich Ataxia: An Impaired Nrf2 Response Mediated via Upregulation of Keap1 and Activation of the Gsk3β Axis

### 24178 ### MAVS Mediates Protection against Myocardial Ischemic Injury with Hydrogen Sulfide David Durrant, Adolfo G Mauro, Juan Valle Raleigh, Anindita Das, Jun He, Khoa Nguyen, Stefano Toldo, Antonio Abbate, Fadi N Salloum; Virginia Commonwealth Univ, Richmond, VA Background : Hydrogen sulfide (H2S) has been shown to protect against myocardial ischemic and inflammatory injury in part by preserving mitochondrial integrity. Since mitochondrial antiviral signaling (MAVS) protein has been implicated in attenuating Bax-mediated cytochrome c release from mitochondria caused by oxidative stress or ischemia, we sought to determine whether MAVS mediates the cardioprotective effects of H2S. Methods and Results : After baseline echocardiography, adult male wild type (WT) or MAVS KO mice underwent myocardial infarction (MI) by coronary artery ligation for 30 min. followed by 24 h reperfusion. Mice were pretreated with Na2S (100 μg/kg; ip ) or saline 1 h before MI. Infarct size, measured with TTC staining, was reduced and LV fractional shortening (FS) was preserved with Na2S at 24h post MI in WT mice as compared to saline-treated mice, but not in MAVS KO mice (Figs. A and B). The risk area was not different between the groups. Western blot analysis revealed a significant decline in myocardial MAVS expression at 24h post MI, which was preserved with Na2S (Fig. C). Another subset of mice was subjected to permanent coronary artery occlusion and treated with Na2S or saline daily for 28 days. LVFS decreased significantly at 28 days post-MI in the saline group, but was significantly preserved with Na2S (Fig. D). Moreover, LV infarct scar size, assessed by trichrome staining, was smaller in Na2S group (22.4 ± 2.7%) as compared to control (33.5 ± 2.1%, P<0.05). Survival rate was 2 fold higher with Na2S compared to saline (P<0.05). Western blot analysis confirmed a significant …

Human Bartter syndrome IV is an autosomal recessive disorder characterized by congenital deafness and severe renal salt and fluid loss. It is caused by mutations in BSND, which encodes barttin, a beta-subunit of ClC-Ka and ClC-Kb chloride channels. Inner-ear-specific disruption of Bsnd in mice now reveals that the positive potential, but not the high potassium concentration, of the scala media depends on the presence of these channels in the epithelium of the stria vascularis. The reduced driving force for K(+)-entry through mechanosensitive channels into sensory hair cells entails a profound congenital hearing loss and subtle vestibular symptoms. Although retaining all cell types and intact tight junctions, the thickness of the stria is reduced early on. Cochlear outer hair cells degenerate over several months. A collapse of endolymphatic space was seen when mice had additionally renal salt and fluid loss due to partial barttin deletion in the kidney. Bsnd(-/-) mice thus demonstrate a novel function of Cl(-) channels in generating the endocochlear potential and reveal the mechanism leading to deafness in human Bartter syndrome IV.

The role of other STAT subtypes in conferring ischemic tolerance is unclear. We hypothesized that in STAT-3 deletion alternative STAT subtypes would protect myocardial function against ischemia-reperfusion injury. Wild-type (WT) male C57BL/6 mice or mice with cardiomyocyte STAT-3 knockout (KO) underwent baseline echocardiography. Langendorff-perfused hearts underwent ischemic preconditioning (IPC) or no IPC before ischemia-reperfusion. Following ex vivo perfusion, hearts were analyzed for STAT-5 and -6 phosphorylation by Western blot analysis of nuclear fractions. Echocardiography and postequilibration cardiac performance revealed no differences in cardiac function between WT and KO hearts. Phosphorylated STAT-5 and -6 expression was similar in WT and KO hearts before perfusion. Contractile function in WT and KO hearts was significantly impaired following ischemia-reperfusion in the absence of IPC. In WT hearts, IPC significantly improved the recovery of the maximum first derivative of developed pressure (+dP/dtmax) compared with that in hearts without IPC. IPC more effectively improved end-reperfusion dP/dtmax in WT hearts compared with KO hearts. Preconditioned and nonpreconditioned KO hearts exhibited increased phosphorylated STAT-5 and -6 expression compared with WT hearts. The increased subtype activation did not improve the efficacy of IPC in KO hearts. In conclusion, baseline cardiac performance is preserved in hearts with cardiac-restricted STAT-3 deletion. STAT-3 deletion attenuates preconditioning and is not associated with a compensatory upregulation of STAT-5 and -6 subtypes. The activation of STAT-5 and -6 in KO hearts following ischemic challenge does not provide functional compensation for the loss of STAT-3. JAK-STAT signaling via STAT-3 is essential for effective IPC.

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the mouse heart. This study used NRTN knockout (KO) and wild type (WT) mice to determine the impact of NRTN deletion on adult intracardiac cholinergic neurons and nerve fibers and on cardiac cholinergic function. Immunohistochemical analysis of atrial tissue stained for vesicular ACh transporter revealed that sinoatrial node cholinergic nerve density was reduced by more than 40% in 8 week KO hearts. Additionally, the number of cholinergic neurons in KO hearts was 80% less than in WT, and KO neurons were 22% smaller in size. Atrial ACh levels were significantly lower in 16 week KO mice compared to WT (1.8±0.3 vs 3.7±0.8 pmol/mg wt; P&lt;0.02), as expected from cholinergic neuron and nerve fiber deficits. Maximum heart rate (HR) responses to right vagal nerve stimulation (VNS) were decreased in 16 week KO (38±6 vs 81±3% decrease at 20Hz; P&lt;0.0001), with no difference in baseline HR. KO animals took longer to reach maximum bradycardia during VNS and longer to recover to baseline HR upon stimulus termination. Data from isolated atrial preparations showed no change in postjunctional cholinergic sensitivity. These results demonstrate that NRTN KO leads to structural and functional cholinergic deficits in the heart but residual cholinergic nerve fibers retain ability to modify cardiac function. Supported by the AHA SE Affiliate.

Cardiac K-ATP channels have been recently been implicated to serve a homeostatic function under catecholamine stress. Since the use of drugs that modulate K-ATP channels is common in clinical practice, the implications of channel function on cardiac electrophysiology warrant further investigation. Here, using simultaneous monophasic action potential and ECG recordings, we examined the arrhythmogenic consequences of genetic deletion of the pore-forming K-ATP channel subunit Kir6.2. Infusion of the sympathomimetic isoproterenol (1muM) readily induced early afterdepolarizations in Kir6.2 knockout (KO) but not in the wildtype (WT) hearts (incidence: 96.8% versus 1%, p<0.01). This translated into an order of 10 magnitude higher incidence of triggered activity and associated premature ventricular contraction in the KO hearts. Although there was no significant difference in perfusion flow between KO and WT hearts before or after isoproterenol infusion, the shortening of the action potential duration at 90% repolarization under sympathomimetic challenge observed in the WT (from 82 to 74 ms, p<0.01) was absent in the KO (79 to 80 ms, p>0.05). We conclude that cardiac K-ATP channels are required to secure proper repolarization reserve and prevent afterdepolarization-associated arrhythmia under sympathetic stress. Therefore, clinical conditions that are associated with reduced K-ATP channel activity, such as use of sulfonylurea drugs, may predispose to arrhythmic events. Clinical Pharmacology & Therapeutics (2004) 75, P48–P48; doi: 10.1016/j.clpt.2003.11.181