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Cardiac Na/K-ATPase (NKA) inhibition by cardiotonic steroids (CTS) modulates intracellular Na+ and Ca2+, leading to increased force of contraction. In contrast, hypertrophy of cardiomyocytes (CM) is triggered by CTS through stimulation of reactive oxygen species (ROS) and fetal gene re-expression independently of changes in intracellular Na+ or Ca2+. Based on recent evidence that NKAα1 serves an isoform-specific function in the regulation of oxidative metabolism and ROS generation, we used a genetic approach in the mouse and in human cardiac cells (AC16) to test the hypothesis that NKAα1 is required for cardiac hypertrophy. Cardio α1-/- mice, a conditional KO of NKAα1 in the heart, were viable without overt abnormality of cardiac structure or function. NKAα1 was undetectable in heart lysates or isolated CM of adult cardio α1-/- mice. Despite a compensatory upregulation of NKAα2, Na/K-ATPase activity in crude heart homogenates and NKA currents in CM were reduced by about 60%. Decreased ROS (assessed by total protein carbonylation) and reduced fatty acid oxidation (FAO) and oxidative phosphorylation pathways (revealed by RNA-seq analysis) were noted at baseline. RT-qPCR revealed downregulated genes involved with FAO and the electron transport chain (such as, Acadl and Ndufa9, about 20% decrease, n=6) in cardio α1-/- hearts. The activity of the electron transport chain complex I was decreased in cardio α1-/- hearts (0.3354 vs 0.2732 mOD/min/mg of protein, p-1.day-1 for 1 week, n=6-15), cardio α1-/- mice did not develop cardiac hypertrophy, as assessed by heart weight to tibia length ratio, but displayed exacerbated cardiac fibrosis, revealed by Trichrome staining, consistent with the postulated role of CM NKAα1 isoform-specific function in cardiac hypertrophy in vivo. In vitro, CRISPR-Cas9-mediated KO of NKAα1 in AC16 cells led to a drastic downregulation of NKAα1 compared to WT AC16 cells. In contrast to WT AC16 cells, NKAα1 KO cells did not respond to the α-adrenergic agonist phenylephrine (50 μM for 24h) with the expected increase in cell area (2243 vs 3520 μm2 for WT, p2 for KO, p>0.05). These data suggest that NKAα1 isoform-specific function is required for cardiac hypertrophy. Mechanistically, our data support an isoform-specific regulatory function of NKAα1 in normal FAO, electron transport chain activity, and ROS generation, which are critical for CM hypertrophy. Supported by NIH Grant R15 HL145666 and American Heart Association Grant 22POST917776 (Marco T. Pessoa) This is the full abstract presented at the American Physiology Summit 2023 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.

Ngkelo et al. use a mast cell–deficient mouse model to reveal a protective role of mast cells in myocardial infarction, through regulation of the cardiac contractile machinery., Acute myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Inflammatory cells orchestrate postischemic cardiac remodeling after MI. Studies using mice with defective mast/stem cell growth factor receptor c-Kit have suggested key roles for mast cells (MCs) in postischemic cardiac remodeling. Because c-Kit mutations affect multiple cell types of both immune and nonimmune origin, we addressed the impact of MCs on cardiac function after MI, using the c-Kit–independent MC-deficient (Cpa3Cre/+) mice. In response to MI, MC progenitors originated primarily from white adipose tissue, infiltrated the heart, and differentiated into mature MCs. MC deficiency led to reduced postischemic cardiac function and depressed cardiomyocyte contractility caused by myofilament Ca2+ desensitization. This effect correlated with increased protein kinase A (PKA) activity and hyperphosphorylation of its targets, troponin I and myosin-binding protein C. MC-specific tryptase was identified to regulate PKA activity in cardiomyocytes via protease-activated receptor 2 proteolysis. This work reveals a novel function for cardiac MCs modulating cardiomyocyte contractility via alteration of PKA-regulated force–Ca2+ interactions in response to MI. Identification of this MC-cardiomyocyte cross-talk provides new insights on the cellular and molecular mechanisms regulating the cardiac contractile machinery and a novel platform for therapeutically addressable regulators.

Rationale: Cardiac tissue cohesion relying on highly ordered cardiomyocytes (CM) interactions is critical because most cardiomyopathies are associated with tissue remodeling and architecture alterations. Objective: Eph/ephrin system constitutes a ubiquitous system coordinating cellular communications which recently emerged as a major regulator in adult organs. We examined if eph/ephrin could participate in cardiac tissue cyto-organization. Methods and Results: We reported the expression of cardiac ephrin-B1 in both endothelial cells and for the first time in CMs where ephrin-B1 localized specifically at the lateral membrane. Ephrin-B1 knock-out (KO) mice progressively developed cardiac tissue disorganization with loss of adult CM rod-shape and sarcomeric and intercalated disk structural disorganization confirmed in CM-specific ephrin-B1 KO mice. CMs lateral membrane exhibited abnormal structure by electron microscopy and notably increased stiffness by atomic force microscopy. In wild-type CMs, ephrin-B1 interacted with claudin-5/ZO-1 complex at the lateral membrane, whereas the complex disappeared in KO/CM-specific ephrin-B1 KO mice. Ephrin-B1 deficiency resulted in decreased mRNA expression of CM basement membrane components and disorganized fibrillar collagen matrix, independently of classical integrin/dystroglycan system. KO/CM-specific ephrin-B1 KO mice exhibited increased left ventricle diameter and delayed atrioventricular conduction. Under pressure overload stress, KO mice were prone to death and exhibited striking tissue disorganization. Finally, failing CMs displayed downregulated ephrin-B1/claudin-5 gene expression linearly related to the ejection fraction. Conclusions: Ephrin-B1 is necessary for cardiac tissue architecture cohesion by stabilizing the adult CM morphology through regulation of its lateral membrane. Because decreased ephrin-B1 is associated with molecular/functional cardiac defects, it could represent a new actor in the transition toward heart failure.

Periodontitis and type 2 diabetes are connected pandemic diseases, and both are risk factors for cardiovascular complications. Nevertheless, the molecular factors relating these two chronic pathologies are poorly understood. We have shown that, in response to a long-term fat-enriched diet, mice present particular gut microbiota profiles related to three metabolic phenotypes: diabetic-resistant (DR), intermediate (Inter), and diabetic-sensitive (DS). Moreover, many studies suggest that a dysbiosis of periodontal microbiota could be associated with the incidence of metabolic and cardiac diseases. We investigated whether periodontitis together with the periodontal microbiota may also be associated with these different cardiometabolic phenotypes. We report that the severity of glucose intolerance is related to the severity of periodontitis and cardiac disorders. In detail, alveolar bone loss was more accentuated in DS than Inter, DR, and normal chow-fed mice. Molecular markers of periodontal inflammation, such as TNF-α and plasminogen activator inhibitor-1 mRNA levels, correlated positively with both alveolar bone loss and glycemic index. Furthermore, the periodontal microbiota of DR mice was dominated by the Streptococcaceae family of the phylum Firmicutes, whereas the periodontal microbiota of DS mice was characterized by increased Porphyromonadaceae and Prevotellaceae families. Moreover, in DS mice the periodontal microbiota was indicated by an abundance of the genera Prevotella and Tannerella, which are major periodontal pathogens. PICRUSt analysis of the periodontal microbiome highlighted that prenyltransferase pathways follow the cardiometabolic adaptation to a high-fat diet. Finally, DS mice displayed a worse cardiac phenotype, percentage of fractional shortening, heart rhythm, and left ventricle weight-to-tibia length ratio than Inter and DR mice. Together, our data show that periodontitis combined with particular periodontal microbiota and microbiome is associated with metabolic adaptation to a high-fat diet related to the severity of cardiometabolic alteration.

Background— Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and is regulated by various signaling pathways. However, the molecular mechanisms that negatively regulate these signal transduction pathways remain poorly understood. Methods and Results— Here, we characterized Carabin, a protein expressed in cardiomyocytes that was downregulated in cardiac hypertrophy and human heart failure. Four weeks after transverse aortic constriction, Carabin-deficient (Carabin −/− ) mice developed exaggerated cardiac hypertrophy and displayed a strong decrease in fractional shortening (14.6±1.6% versus 27.6±1.4% in wild type plus transverse aortic constriction mice; P P 2+ /calmodulin-dependent protein kinase II activation and prevented nuclear export of histone deacetylase 4 after adrenergic stimulation or myocardial pressure overload. Finally, we showed that Carabin Ras–GTPase–activating protein domain and calcineurin-interacting domain were both involved in the antihypertrophic action of Carabin. Conclusions— Our study identifies Carabin as a negative regulator of key prohypertrophic signaling molecules, calcineurin, Ras, and Ca 2+ /calmodulin-dependent protein kinase II and implicates Carabin in the development of cardiac hypertrophy and failure.

Inflammatory cells orchestrate post-ischemic cardiac remodeling after myocardial infarction (MI). Studies in Kit mutant mice suggest key roles for mast cells in post-ischemic tissue remodeling. However, Kit mutations affect multiple cell types of both immune and non-immune origin. The aim of this study was to address the impact of mast cells on cardiac function following MI, using selectively mast cell-deficient mice (Cpa3Cre/+ mice).The number of mast cell progenitor (Lin-CD45+CD34+FcγRII/III+β7+) increased in the bone marrow and the white adipose tissue on day 3 after MI, infiltrated the heart on day 5 and differentiated to mature mast cells (Cd117+FcεRI+Sca1+) by day 7 in cardiac tissue. To assess the functional effect of mast cell infiltration, the cardiac function 14 days following MI was assessed in wild-type (WT) and 3 mast cells deficient mice models, the kitW/Wv mice, the Cpa3Cre/+ mice and mice treated with the degranulation inhibitor DSCG (50 μg/g, once a day). There was a significant decrease of the left ventricular shortening fraction in all 3 mast cell deficient models compared to WT mice (1.4-, 1.6- and 2-fold respectively) that correlated with increased cardiomyocyte apoptosis in the kitW/Wv mice; and increased fibrosis in the DSCG-treated mice (n=8-14 per group, p