Double Knockout Hearts Research Articles

Abstract MP161: A Calcineurin-hoxb13 Axis Regulates Growth Mode of Mammalian Cardiomyocytes

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. Our group recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2. We recently also showed that Meis1, a TALE family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a Meis1 cofactor in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can both extend the postnatal window of cardiomyocyte proliferation and reactivate cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1/Hoxb13 double knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and an improvement in left ventricular systolic function following myocardial infarction both by echocardiography and MRI. ChIP-seq analysis demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204 (S204), resulting in its nuclear localization and cell cycle arrest. Collectively, these results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

Exacerbation of dystrophic cardiomyopathy by phospholamban deficiency mediated chronically increased cardiac Ca2+ cycling in vivo.

Cardiomyopathy is a significant contributor to morbidity and mortality in Duchenne muscular dystrophy (DMD). Membrane instability, leading to intracellular Ca2+ mishandling and overload, causes myocyte death and subsequent fibrosis in DMD cardiomyopathy. On a cellular level, cardiac myocytes from mdx mice have dysregulated Ca2+ handling, including increased resting Ca2+ and slow Ca2+ decay, especially evident under stress conditions. Sarco(endo)plasmic reticulum Ca2+ ATPase and its regulatory protein phospholamban (PLN) are potential therapeutic targets for DMD cardiomyopathy owing to their key role in regulating intracellular Ca2+ cycling. We tested the hypothesis that enhanced cardiac Ca2+ cycling would remediate cardiomyopathy caused by dystrophin deficiency. We used a genetic complementation model approach by crossing dystrophin-deficient mdx mice with PLN knockout (PLNKO) mice [termed double-knockout (DKO) mice]. As expected, adult cardiac myocytes isolated from DKO mice exhibited increased contractility and faster relaxation associated with increased Ca2+ transient peak height and faster Ca2+ decay rate compared with control mice. However, compared with wild-type, mdx, and PLNKO mice, DKO mice unexpectedly had reduced in vivo systolic and diastolic function as measured by echocardiography. Furthermore, Evans blue dye uptake was increased in DKO hearts compared with control, mdx, and PLNKO hearts, demonstrating increased membrane damage, which subsequently led to increased fibrosis in the DKO myocardium in vivo. In conclusion, despite enhanced intracellular Ca2+ handling at the myocyte level, DMD cardiomyopathy was exacerbated owing to unregulated chronic increases in Ca2+ cycling in DKO mice in vivo. These findings have potentially important implications for ongoing therapeutic strategies for the dystrophic heart. NEW & NOTEWORTHY This study examined the effects of phospholamban ablation on the pathophysiology of cardiomyopathy in dystrophin-deficient mice. In this setting, contractility and Ca2+ cycling were enhanced in isolated myocytes; however, in vivo heart function was diminished. Additionally, sarcolemmal integrity was compromised and fibrosis was increased. This is the first study, to our knowledge, examining unregulated Ca2+ cycling in the dystrophin-deficient heart. Results from this study have implications for potential therapies targeting Ca2+ handling in dystrophic cardiomyopathy. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/unregulated-ca2-cycling-exacerbates-dmd-cardiomyopathy/ .

Abstract 340: Cardiomyocyte-Derived Mechanical Signals Function in a Paracrine Manner to Regulate Tissue Stiffness and Myocardial Proliferation

Cells sense and transduce mechanical signals through cell-cell adhesions and cell-extracellular matrix adhesions. After birth, contractile force generation and tissue stiffness increase as cardiac output increases to support the needs of the newborn organism. During this postnatal period, the N-cadherin adhesion complex assembles at the intercalated disc (ID) as integrin-fibronectin (FN) adhesions decrease, accompanying the switch from hyperplastic to hypertrophic growth. However, little is known regarding the reciprocity between integrin and N-cadherin adhesions in the regulation of cell cycle withdrawal that occurs in cardiomyocytes after birth. α-catenins function as mechanosensors and transduce the intercellular force from N-cadherin to the actin cytoskeleton. Cardiac-specific αE- and αT-catenins double knockout (DKO) mice exhibit sustained cardiomyocyte proliferation beyond the first week of life. To investigate the role of cell-matrix interactions in DKO hearts, the spatial distribution of integrin-FN complexes was examined at postnatal day (P) 4, P7, P14, and P60. DKO hearts exhibited aberrant N-cadherin localization accompanied by altered distribution of α5/β1 integrin, the primary FN receptor. Normally found at the lateral membrane, α5 and β1 accumulated at the ID while N-cadherin was reduced at the termini. FN matrix assembly, as monitored by immunofluorescent staining and its insolubility in the detergent deoxycholate, was increased in DKO hearts. Meanwhile, focal adhesion kinase (FAK) was activated in early postnatal DKO hearts. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated proliferation was dependent on FAK activity. Finally, treatment of DKO pups with the lysyl oxidase inhibitor, β-aminopropionitrile (BAPN), was sufficient to reduce cardiomyocyte proliferation to wild-type levels suggesting that matrix assembly and tissue stiffness feedback to promote cardiomyocyte proliferation in DKO hearts. This data demonstrates that α-catenins regulate the balance between cell-cell and cell-matrix adhesions, which, in turn, controls cardiomyocyte proliferation, thus providing a molecular explanation for loss of regenerative potential shortly after birth.

Glucose is preferentially utilized for biomass synthesis in pressure-overloaded hearts: evidence from fatty acid-binding protein-4 and -5 knockout mice.

AimsThe metabolism of the failing heart is characterized by an increase in glucose uptake with reduced fatty acid (FA) oxidation. We previously found that the genetic deletion of FA-binding protein-4 and -5 [double knockout (DKO)] induces an increased myocardial reliance on glucose with decreased FA uptake in mice. However, whether this fuel switch confers functional benefit during the hypertrophic response remains open to debate. To address this question, we investigated the contractile function and metabolic profile of DKO hearts subjected to pressure overload.Methods and resultsTransverse aortic constriction (TAC) significantly reduced cardiac contraction in DKO mice (DKO-TAC), although an increase in cardiac mass and interstitial fibrosis was comparable with wild-type TAC (WT-TAC). DKO-TAC hearts exhibited enhanced glucose uptake by 8-fold compared with WT-TAC. Metabolic profiling and isotopomer analysis revealed that the pool size in the TCA cycle and the level of phosphocreatine were significantly reduced in DKO-TAC hearts, despite a marked increase in glycolytic flux. The ingestion of a diet enriched in medium-chain FAs restored cardiac contractile dysfunction in DKO-TAC hearts. The de novo synthesis of amino acids as well as FA from glycolytic flux was unlikely to be suppressed, despite a reduction in each precursor. The pentose phosphate pathway was also facilitated, which led to the increased production of a coenzyme for lipogenesis and a precursor for nucleotide synthesis. These findings suggest that reduced FA utilization is not sufficiently compensated by a robust increase in glucose uptake when the energy demand is elevated. Glucose utilization for sustained biomass synthesis further enhances diminishment of the pool size in the TCA cycle.ConclusionsOur data suggest that glucose is preferentially utilized for biomass synthesis rather than ATP production during pressure-overload-induced cardiac hypertrophy and that the efficient supplementation of energy substrates may restore cardiac dysfunction caused by energy insufficiency.

Abstract 80: Interplay Between Cell-cell and Cell-extracellular Matrix Forces Regulate Myocardial Proliferation

Changes in expression and distribution of integrin, fibronectin (FN), and N-cadherin in the postnatal heart accompany the switch from hyperplastic to hypertrophic growth. As FN decreases after birth, N-cadherin/catenin complex accumulates at the cell termini creating a specialized type of cell-cell contact called the intercalated disc (ID). Integrin-FN interactions promote cardiomyocyte and cardiac progenitor cell proliferation. However, little is known regarding the reciprocity between integrin and N-cadherin adhesions in the regulation of myocardial proliferation. Alpha-catenins function as mechanosensors and transduce the intercellular force from N-cadherin to the actin cytoskeleton. To investigate mechanotransduction in the heart, we generated cardiac-specific αE- and αT-catenins double knockout (DKO) mice. The relationship between N-cadherin, integrin, and FN was examined at postnatal day (P) 4, P7, P14, and P60. DKO hearts exhibited aberrant N-cadherin expression accompanied by increased expression of α5/β1 integrin, the primary receptor for FN. Normally found at the lateral membrane, α5 and FN accumulated at the ID along with N-cadherin in DKO hearts. FN-integrin binding leads to the formation of FN fibrils that are initially soluble in the detergent deoxycholate (DOC) but are gradually converted into a stable, DOC-insoluble form that comprises the mature matrix. Both α5 and FN were increased in the DOC-insoluble fraction consistent with enhanced matrix assembly in DKO hearts. Activation of focal adhesion kinase (FAK) and p130CAS were observed in the DKO hearts consistent with increased cell-extracellular matrix interactions. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated Yap nuclear accumulation was dependent on FAK activity. These data demonstrate that α-catenins regulate the balance between cell-cell and cell-matrix adhesions, which, in turn, controls Yap subcellular localization, thus providing a molecular explanation for loss of regenerative potential in the adult heart.

Abstract 13093: An Imbalance in Cardiomyocyte Glucocorticoid and Mineralocorticoid Receptor Signaling Leads to Heart Failure

Stress is increasingly associated with adverse cardiac outcomes; however the mechanisms underlying these effects are poorly understood. Glucocorticoids are primary stress hormones that regulate cell and tissue homeostasis through two closely related nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Cardiomyocytes express both these receptors but little is known concerning their coordinated actions in heart physiology and whether an imbalance in their signaling contributes to cardiac disease. Here, we show that patients with failing hearts have a disparity in the relative levels of GR and MR compared to non-failing donor hearts. To examine the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte-specific deletion of GR (cardioGRKO), MR (cardioMRKO), or both GR and MR (cardioGRMRdKO). The cardioMRKO mice exhibited normal heart function whereas the cardioGRKO mice spontaneously developed cardiac hypertrophy and left ventricular systolic dysfunction. Surprisingly, the cardioGRMRdKO mice were protected from the cardiac disease observed for the cardioGRKO mice. At a molecular level, pathological gene changes present in the GR deficient hearts were also found in the double knockout hearts whereas specific cardioprotective gene changes were detected only in hearts from the cardioGRMRdKO mice. Re-installation of MR into the cardioGRMRdKO hearts by adeno-associated virus gene delivery reversed these cardioprotective gene changes and resulted in cardiac dysfunction. These findings reveal critical gene-regulatory roles for both GR and MR in the heart and suggest that an imbalance in these two signaling pathways leads to heart disease. Therapies designed to modulate cardiomyocyte glucocorticoid signaling to favor more GR and less MR activity may provide new approaches for treating the failing heart.

Myocardin-related transcription factors are required for cardiac development and function

Myocardin-Related Transcription Factors A and B (MRTF-A and MRTF-B) are highly homologous proteins that function as powerful coactivators of serum response factor (SRF), a ubiquitously expressed transcription factor essential for cardiac development. The SRF/MRTF complex binds to CArG boxes found in the control regions of genes that regulate cytoskeletal dynamics and muscle contraction, among other processes. While SRF is required for heart development and function, the role of MRTFs in the developing or adult heart has not been explored. Through cardiac-specific deletion of MRTF alleles in mice, we show that either MRTF-A or MRTF-B is dispensable for cardiac development and function, whereas deletion of both MRTF-A and MRTF-B causes a spectrum of structural and functional cardiac abnormalities. Defects observed in MRTF-A/B null mice ranged from reduced cardiac contractility and adult onset heart failure to neonatal lethality accompanied by sarcomere disarray. RNA-seq analysis on neonatal hearts identified the most altered pathways in MRTF double knockout hearts as being involved in cytoskeletal organization. Together, these findings demonstrate redundant but essential roles of the MRTFs in maintenance of cardiac structure and function and as indispensible links in cardiac cytoskeletal gene regulatory networks.

Alpha-catenins control cardiomyocyte proliferation by regulating Yap activity.

Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. Thus, they are unable to effectively replace dying cells in the injured heart. The recent discovery that the transcriptional coactivator Yes-associated protein (Yap) is necessary and sufficient for cardiomyocyte proliferation has gained considerable attention. However, the upstream regulators and signaling pathways that control Yap activity in the heart are poorly understood. To investigate the role of α-catenins in the heart using cardiac-specific αE- and αT-catenin double knockout mice. We used 2 cardiac-specific Cre transgenes to delete both αE-catenin (Ctnna1) and αT-catenin (Ctnna3) genes either in the perinatal or in the adult heart. Perinatal depletion of α-catenins increased cardiomyocyte number in the postnatal heart. Increased nuclear Yap and the cell cycle regulator cyclin D1 accompanied cardiomyocyte proliferation in the α-catenin double knockout hearts. Fetal genes were increased in the α-catenin double knockout hearts indicating a less mature cardiac gene expression profile. Knockdown of α-catenins in neonatal rat cardiomyocytes also resulted in increased proliferation, which could be blocked by knockdown of Yap. Finally, inactivation of α-catenins in the adult heart using an inducible Cre led to increased nuclear Yap and cardiomyocyte proliferation and improved contractility after myocardial infarction. These studies demonstrate that α-catenins are critical regulators of Yap, a transcriptional coactivator essential for cardiomyocyte proliferation. Furthermore, we provide proof of concept that inhibiting α-catenins might be a useful strategy to promote myocardial regeneration after injury.

Capillary Endothelial Fatty Acid Binding Proteins 4 and 5 Play a Critical Role in Fatty Acid Uptake in Heart and Skeletal Muscle

Fatty acids (FAs) are the major substrate for energy production in the heart. Here, we hypothesize that capillary endothelial fatty acid binding protein 4 (FABP4) and FABP5 play an important role in providing sufficient FAs to the myocardium. Both FABP4/5 were abundantly expressed in capillary endothelium in the heart and skeletal muscle. The uptake of a FA analogue, 125I-15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid, was significantly reduced in these tissues in double-knockout (DKO) mice for FABP4/5 compared with wild-type mice. In contrast, the uptake of a glucose analogue, 18F-fluorodeoxyglucose, was remarkably increased in DKO mice. The expression of transcripts for the oxidative catabolism of FAs was reduced during fasting, whereas transcripts for the glycolytic pathway were not altered in DKO hearts. Notably, metabolome analysis revealed that phosphocreatine and ADP levels were significantly lower in DKO hearts, whereas ATP content was kept at a normal level. The protein expression levels of the glucose transporter Glut4 and the phosphorylated form of phosphofructokinase-2 were increased in DKO hearts, whereas the phosphorylation of insulin receptor-β and Akt was comparable between wild-type and DKO hearts during fasting, suggesting that a dramatic increase in glucose usage during fasting is insulin independent and is at least partly attributed to the post-transcriptional and allosteric regulation of key proteins that regulate glucose uptake and glycolysis. Capillary endothelial FABP4/5 are required for FA transport into FA-consuming tissues that include the heart. These findings identify FABP4/5 as promising targets for controlling the metabolism of energy substrates in FA-consuming organs that have muscle-type continuous capillary.

IL17A and IFNγ jointly control rapidly fatal eosinophilic myocarditis in mice (P4120)

Abstract Deviation to autoaggressive T helper 17 (Th17) responses has been reported to account for the protective effect of interferon-gamma (IFNγ) in certain animal models of autoimmune disease. Building on our previous findings that interleukin-17 (IL17) signaling is critical for the progression of experimental autoimmune myocarditis (EAM) to dilative heart failure, we sought to address the question of whether IL17 mediated the severe form of EAM observed in IFNγ-deficient mice. To our surprise, we found that IFNγ-/-IL17A-/- double-knockout (DKO) mice had greater mortality compared to IFNγ-/- mice, starting at day 14 and reaching 50% by day 21 of EAM. DKO hearts were severely infiltrated, indicating acute inflammatory heart failure as the mode of death in these mice. We found pronounced eosinophilic infiltration in DKO mice, implicating eosinophils as mediators of the enhanced morbidity. Cardiac production of CCL11/eotaxin was increased, alongside Th2 deviation among heart-infiltrating CD4+ T cells. Intriguingly, blockade of IL4 in IFNγ-/-IL17A-/-DKO mice inhibited Th2 deviation, but failed to resolve eosinophilia or morbidity. However, developmental ablation of eosinophils partly rescued the survival of DKO mice. These data point to a novel collaboration between IFNγ and IL17A in controlling eosinophil recruitment.

The Xin repeat-containing protein, mXinβ, initiates the maturation of the intercalated discs during postnatal heart development

The intercalated disc (ICD) is a unique structure to the heart and plays vital roles in communication and signaling among cardiomyocytes. ICDs are formed and matured during postnatal development through a profound redistribution of the intercellular junctions, as well as recruitment and assembly of more than 200 proteins at the termini of cardiomyocytes. The molecular mechanism underlying this process is not completely understood. The mouse orthologs (mXinα and mXinβ) of human cardiomyopathy-associated (CMYA)/Xin actin-binding repeat-containing protein (XIRP) genes (CMYA1/XIRP1 and CMYA3/XIRP2, respectively) encode proteins localized to ICDs. Ablation of mXinα results in adult late-onset cardiomyopathy with conduction defects and up-regulation of mXinβ. ICD structural defects are found in adult but not juvenile mXinα-null hearts. On the other hand, loss of mXinβ leads to ICD defects at postnatal day 16.5, a developmental stage when the heart is forming ICDs, suggesting mXinβ is required for ICD formation. Using quantitative Western blot, we showed in this study that mXinβ but not mXinα was uniquely up-regulated during the redistribution of intercellular junction from the lateral membrane of cardiomyocytes to their termini. In the absence of mXinβ, the intercellular junctions failed to be restricted to the termini of the cells, and the onset of such defect correlated with the peak expression of mXinβ. Immunofluorescence staining and subcellular fractionation showed that mXinβ preferentially associated with the forming ICDs, further suggesting that mXinβ functioned locally to promote ICD maturation. In contrast, the spatiotemporal expression profile of mXinα and the lack of more severe ICD defects in mXinα−/−;mXinβ−/− double knockout hearts than in mXinβ−/− hearts suggested that mXinα was not essential for the postnatal formation of ICDs. A two-step model for the development of ICD is proposed where mXinβ is essential for the redistribution of intercellular junction components from the lateral puncta to the cell termini.

Mitofusins 1 and 2 Are Essential for Postnatal Metabolic Remodeling in Heart

At birth, there is a switch from placental to pulmonary circulation and the heart commences its aerobic metabolism. In cardiac myocytes, this transition is marked by increased mitochondrial biogenesis and remodeling of the intracellular architecture. The mechanisms governing the formation of new mitochondria and their expansion within myocytes remain largely unknown. Mitofusins (Mfn-1 and Mfn-2) are known regulators of mitochondrial networks, but their role during perinatal maturation of the heart has yet to be examined. The objective of this study was to determine the significance of mitofusins during early postnatal cardiac development. We genetically inactivated Mfn-1 and Mfn-2 in midgestational and postnatal cardiac myocytes using a loxP/Myh6-cre approach. At birth, cardiac morphology and function of double-knockout (DKO) mice are normal. At that time, DKO mitochondria increase in numbers, appear to be spherical and heterogeneous in size, but exhibit normal electron density. By postnatal day 7, the mitochondrial numbers in DKO myocytes remain abnormally expanded and many lose matrix components and membrane organization. At this time point, DKO mice have developed cardiomyopathy. This leads to a rapid decline in survival and all DKO mice die before 16 days of age. Gene expression analysis of DKO hearts shows that mitochondria biogenesis genes are downregulated, the mitochondrial DNA is reduced, and mitochondrially encoded transcripts and proteins are also reduced. Furthermore, mitochondrial turnover pathways are dysregulated. Our findings establish that Mfn-1 and Mfn-2 are essential in mediating mitochondrial remodeling during postnatal cardiac development, a time of dramatic transitions in the bioenergetics and growth of the heart.

IL17A switches from a pathogenic to a protective function in experimental autoimmune myocarditis in the absence of IFNγ. (115.14)

Abstract Deviation to autoaggressive Th17 responses has been reported to account for the protective effect of IFNγ in certain animal models of autoimmune disease. Building on our previous findings that IL17 signaling was critical for the progression of experimental autoimmune myocarditis (EAM) to dilative heart failure, we sought to address the question of whether IL17 mediated the severe form of EAM observed in IFNγ-deficient mice. To our surprise, we found that IFNγ-/-IL17A-/- double-knockout (DKO) mice had greater mortality compared to IFNγ-/- mice, starting at day 14 and reaching 50% by day 21 of EAM. DKO hearts were severely infiltrated, indicating acute inflammatory heart failure as the mode of death in these mice. We have found evidence of aberrant eosinophilic Th2-deviated infiltration in DKO mice, implicating eosinophils in mediating the enhanced morbidity. Production of IL5 by CD4+ cells was increased in DKO hearts, as well as cardiac production of CCL11/eotaxin. Heart-infiltrating DKO CD4+ cells showed evidence of Th2 deviation. These data point to a novel collaboration between IFNγ and IL17A in controlling eosinophil recruitment by autoaggressive Th2 cells. Moreover, they indicate that myocarditis is not restricted to a single CD4+ subset - but instead reflects redundancy among Th1, Th2, and Th17 populations to elicit qualititative and quantitative variations in disease phenotype and progression.

Angiotensin II Type 1a Receptor Signals are Involved in the Progression of Heart Failure in MLP-Deficient Mice

Angiotensin II (AT) is implicated in the development of cardiac remodeling, which leads to heart failure, and pharmacological inhibition of the AT type 1 (AT1) receptor has improved mortality and morbidity in patients of heart failure. The aim of this study was to elucidate the role of the AT1 receptor in disease progression in muscle LIM protein (MLP)-deficient mice, which are susceptible to heart failure because of defective function of mechanosensors in cardiomyocytes. Hearts from MLP knockout (MLPKO) mice and MLP-AT1a receptor double knockout (DKO) mice were analyzed. MLPKO hearts showed marked chamber dilatation with cardiac fibrosis and reactivation of the fetal gene program. All of these changes were significantly milder in the DKO hearts. Impaired left ventricular (LV) contractility and filling were alleviated in DKO hearts. However, the impaired relaxation and downregulated expression of sarcoplasmic reticulum calcium-ATPase 2 were unchanged in DKO hearts. The AT1a receptor is involved in progression of LV remodeling and deterioration of cardiac function in the hearts of MLPKO mice. These results suggest that blockade of the receptor is effective in preventing progression of heart failure in dilated cardiomyopathy.