Pressure Overload-induced Cardiac Dysfunction Research Articles

Therapeutic Inhibition of PHF21B Attenuates Pathological Cardiac Hypertrophy by Inhibiting the BMP4/GSK3β/β-Catenin Axis.

Pathological cardiac hypertrophy is a hallmark of various cardiovascular diseases, unfortunately, effective targeted therapies are still lacking. This study aims to verify the role of plant-homeodomain finger protein21b (PHF21B) in pathological cardiac hypertrophy. Angiotensin-II (Ang II) induced cardiomyocyte hypertrophy in vitro, and short hairpin (sh) RNA-mediated PHF21B silencing was used to assess its role in hypertrophic growth. Transverse aortic constriction (TAC) was performed to induce cardiac hypertrophy in mice. To assess the effect of PHF21B on pathological cardiac hypertrophy in vivo, the myocardium was transduced with adeno-associated virus 9 (AAV9) encoding a PHF21B-targeting shRNA for gene ablation. Chromatin immunoprecipitation-polymerase chain reaction (PCR), western blotting, and quantitative reverse transcription-PCR were performed to elucidate the mechanisms through which PHF21B regulates pathological cardiac hypertrophy. This investigation revealed that PHF21B levels were elevated in patients with pathological cardiac hypertrophy. PHF21B inhibition alleviated pressure overload-induced cardiac dysfunction and hypertrophy in vivo, and Ang-II-induced cardiomyocyte hypertrophy in vitro. Genome-wide transcriptome analysis and biological experiments revealed that PHF21B silencing inhibited the Wnt signalling pathway, include the protein expression of β-catenin, and the phosphorylation of glycogen synthase kinase (GSK)-3β. Mechanistically, PHF21B influenced the translation of bone morphogenetic protein (BMP)-4 and facilitated the activation of the GSK3β/β-catenin pathway. The anti-hypertrophic effects of PHF21B knockdown were blocked by BMP4 supplementation. Collectively, our results demonstrated that PHF21B is contributes to pathological cardiac hypertrophy by regulating BMP4 expression and the GSK3β/β-catenin pathway. The inhibition of PHF21B is a potential new therapeutic strategy to mitigate pathological cardiiac hypertrophy.

NRF2 activation in the heart induces glucose metabolic reprogramming and reduces cardiac dysfunction via upregulation of the pentose phosphate pathway.

The transcription factor NRF2 is well recognized as a master regulator of antioxidant responses and cytoprotective genes. Previous studies showed that NRF2 enhances resistance of mouse hearts to chronic hemodynamic overload at least in part by reducing oxidative stress. Evidence from other tissues suggests that NRF2 may modulate glucose intermediary metabolism but whether NRF2 has such effects in the heart is unclear. Here, we investigate the role of NRF2 in regulating glucose intermediary metabolism and cardiac function during disease stress. Cardiomyocyte-specific Keap1 knockout (csKeap1KO) mice, deficient in the endogenous inhibitor of NRF2, were used as a novel model of constitutively active NRF2 signaling. Targeted metabolomics and isotopomer analysis were employed in studies with 13C6-glucose in csKeap1KO and wild-type (WT) mice. Pharmacological and genetic approaches were utilized in neonatal rat ventricular cardiomyocytes (NRVM) to explore molecular mechanisms. We found that cardiac-specific activation of NRF2 redirected glucose metabolism towards the pentose phosphate pathway (PPP), a branch pathway of glycolysis, and mitigated pressure overload-induced cardiomyocyte death and cardiac dysfunction. Activation of NRF2 also protected against myocardial infarction-induced DNA damage in remote myocardium and cardiac dysfunction. In vitro, knockdown of Keap1 upregulated PPP enzymes and reduced cell death in NRVM subjected to chronic neurohumoral stimulation. These pro-survival effects were abolished by pharmacological inhibition of the PPP or silencing of the PPP rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD). Knockdown of NRF2 in NRVM increased stress-induced DNA damage which was rescued by supplementing the cells with either NADPH or nucleosides, the two main products of the PPP. These results indicate that NRF2 regulates cardiac metabolic reprogramming by stimulating the diversion of glucose into the PPP, thereby generating NADPH and providing nucleotides to prevent stress-induced DNA damage and cardiac dysfunction.

Myoferlin alleviates pressure overload-induced cardiac hypertrophy and dysfunction by inhibiting NLRP3-mediated pyroptosis.

Myoferlin (MYOF) is a muscle-derived secretory protein. Recent studies have found that MYOF protects against cell damage. However, the role of MYOF in cardiac hypertrophy remains unclear. Increasing evidence suggests that NLRP3 (NOD-like receptor protein 3) and the pyroptosis cascade play critical roles in the development of cardiac hypertrophy and inflammation. To investigate the role of MYOF in cardiac hypertrophy, we conducted a transverse aortic constriction (TAC) experiment in a mouse model. We found that MYOF can improve cardiac hypertrophy and cardiac function. Furthermore, our study confirmed a connection between cardiac hypertrophy and myocardial pyroptosis. Cardiac hypertrophy significantly increased the proportion of apoptotic cells and upregulated apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and gasdermin D (GSDMD). This suggests that pharmacological or genetic inhibition of NLRP3 can effectively reduce cardiac hypertrophy. An abnormal increase in NLRP3 can reverse the cardioprotective effects of MYOF. Our findings indicate that MYOF is a potential therapeutic agent for cardiac hypertrophy.

IRS2 Signaling Protects Against Stress-Induced Arrhythmia by Maintaining Ca2+ Homeostasis.

The docking protein IRS2 (insulin receptor substrate protein-2) is an important mediator of insulin signaling and may also regulate other signaling pathways. Murine hearts with cardiomyocyte-restricted deletion of Irs2 (cIRS2-KO) are more susceptible to pressure overload-induced cardiac dysfunction, implying a critical protective role of IRS2 in cardiac adaptation to stress through mechanisms that are not fully understood. There is limited evidence regarding the function of IRS2 beyond metabolic homeostasis regulation, particularly in the context of cardiac disease. A retrospective analysis of an electronic medical record database was conducted to identify patients with IRS2 variants and assess their risk of cardiac arrhythmias. Arrhythmia susceptibility was examined in cIRS2-KO mice. The underlying mechanisms were investigated using confocal calcium imaging of ex vivo whole hearts and isolated cardiomyocytes to assess calcium handling, Western blotting to analyze the involved signaling pathways, and pharmacological and genetic interventions to rescue arrhythmias in cIRS2-KO mice. The retrospective analysis identified patients with IRS2 variants of uncertain significance with a potential association to an increased risk of cardiac arrhythmias compared with matched controls. cIRS2-KO hearts were found to be prone to catecholamine-sensitive ventricular tachycardia and reperfusion ventricular tachycardia. Confocal calcium imaging of ex vivo whole hearts and single isolated cardiomyocytes from cIRS2-KO hearts revealed decreased Ca²⁺ transient amplitudes, increased spontaneous Ca²⁺ sparks, and reduced sarcoplasmic reticulum Ca²⁺ content during sympathetic stress, indicating sarcoplasmic reticulum dysfunction. We identified that overactivation of the AKT1/NOS3 (nitric oxide synthase 3)/CaMKII (Ca²⁺/calmodulin-dependent protein kinase II)/RyR2 (type 2 ryanodine receptor) signaling pathway led to calcium mishandling and catecholamine-sensitive ventricular tachycardia in cIRS2-KO hearts. Pharmacological AKT inhibition or genetic stabilization of RyR2 rescued catecholamine-sensitive ventricular tachycardia in cIRS2-KO mice. Cardiac IRS2 inhibits sympathetic stress-induced AKT/NOS3/CaMKII/RyR2 overactivation and calcium-dependent arrhythmogenesis. This novel IRS2 signaling axis, essential for maintaining cardiac calcium homeostasis under stress, presents a promising target for developing new antiarrhythmic therapies.

Blocking CTLA-4 promotes pressure overload-induced heart failure via activating Th17 cells.

Targeting cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) with specific antibody offers long-term benefits for cancer immunotherapy but can cause severe adverse effects in the heart. This study aimed to investigate the role of anti-CTLA-4 antibody in pressure overload-induced cardiac remodeling and dysfunction. Transverse aortic constriction (TAC) was used to induce cardiac hypertrophy and heart failure in mice. Two weeks after the TAC treatment, mice received anti-CTLA-4 antibody injection twice a week at a dose of 10 mg/kg body weight. The administration of anti-CTLA-4 antibody exacerbated TAC-induced decline in cardiac function, intensifying myocardial hypertrophy and fibrosis. Further investigation revealed that anti-CTLA-4 antibody significantly elevated systemic inflammatory factors levels and facilitated the differentiation of T helper 17 (Th17) cells in the peripheral blood of TAC-treated mice. Importantly, anti-CTLA-4 mediated differentiation of Th17 cells and hypertrophic phenotype in TAC mice were dramatically alleviated by the inhibition of interleukin-17A (IL-17A) by an anti-IL-17A antibody. Furthermore, the C-X-C motif chemokine receptor 4 (CXCR4) antagonist AMD3100, also reversed anti-CTLA-4-mediated cardiotoxicity in TAC mice. Overall, these results suggest that the administration of anti-CTLA-4 antibody exacerbates pressure overload-induced heart failure by activating and promoting the differentiation of Th17 cells. Targeting the CXCR4/Th17/IL-17A axis could be a potential therapeutic strategy for mitigating immune checkpoint inhibitors-induced cardiotoxicity.

Cardiomyocyte-specific RXFP1 overexpression protects against pressure overload-induced cardiac dysfunction independently of relaxin

Heart failure (HF) prevalence is rising due to reduced early mortality and demographic change. Relaxin (RLN) mediates protective effects in the cardiovascular system through Relaxin-receptor 1 (RXFP1). Cardiac overexpression of RXFP1 with additional RLN supplementation attenuated HF in the pressure-overload transverse aortic constriction (TAC) model. Here, we hypothesized that robust transgenic RXFP1 overexpression in cardiomyocytes (CM) protects from TAC-induced HF even in the absence of RLN.Hence, transgenic mice with a CM-specific overexpression of human RXFP1 (hRXFP1tg) were generated. Receptor functionality was demonstrated by in vivo hemodynamics, where the administration of RLN induced positive inotropy strictly in hRXFP1tg. An increase in phospholamban-phosphorylation at serine 16 was identified as a molecular correlate. hRXFP1tg were protected from TAC without additional RLN administration, presenting not only less decline in systolic left ventricular (LV) function but also abrogated LV dilation and pulmonary congestion compared to WT mice. Molecularly, transgenic hearts exhibited not only a significantly attenuated fetal and fibrotic gene activation but also demonstrated less fibrotic tissue and CM hypertrophy in histological sections. These protective effects were evident in both sexes. Similar cardioprotective effects of hRXFP1tg were detectable in a RLN-knockout model, suggesting an alternative mechanism of receptor activation through intrinsic activity, alternative endogenous ligands or crosstalk with other receptors.In summary, CM-specific RXFP1 overexpression provides protection against TAC even in the absence of endogenous RLN. This suggests RXFP1 overexpression as a potential therapeutic approach for HF, offering baseline protection with optional RLN supplementation for specific activation.

Septin4 Promotes Fibrosis In The Heart After Pressure Overload

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.

Fibroblast-specific PRMT5 deficiency suppresses cardiac fibrosis and left ventricular dysfunction in male mice

Protein arginine methyltransferase 5 (PRMT5) is a well-known epigenetic regulatory enzyme. However, the role of PRMT5-mediated arginine methylation in gene transcription related to cardiac fibrosis is unknown. Here we show that fibroblast-specific deletion of PRMT5 significantly reduces pressure overload-induced cardiac fibrosis and improves cardiac dysfunction in male mice. Both the PRMT5-selective inhibitor EPZ015666 and knockdown of PRMT5 suppress α-smooth muscle actin (α-SMA) expression induced by transforming growth factor-β (TGF-β) in cultured cardiac fibroblasts. TGF-β stimulation promotes the recruitment of the PRMT5/Smad3 complex to the promoter site of α-SMA. It also increases PRMT5-mediated H3R2 symmetric dimethylation, and this increase is inhibited by Smad3 knockdown. TGF-β stimulation increases H3K4 tri-methylation mediated by the WDR5/MLL1 methyltransferase complex, which recognizes H3R2 dimethylation. Finally, treatment with EPZ015666 significantly improves pressure overload-induced cardiac fibrosis and dysfunction. These findings suggest that PRMT5 regulates TGF-β/Smad3-dependent fibrotic gene transcription, possibly through histone methylation crosstalk, and plays a critical role in cardiac fibrosis and dysfunction.

Kielin/chordin-like protein deficiency aggravates pressure overload-induced cardiac dysfunction and remodeling via P53/P21/CCNB1 signaling in mice.

Targeting cardiac remodeling is regarded as a key therapeutic strategy for heart failure. Kielin/chordin-like protein (KCP) is a secretory protein with 18 cysteine-rich domains and associated with kidney and liver fibrosis. However, the relationship between KCP and cardiac remodeling remains unclear. Here, we aimed to investigate the role of KCP in cardiac remodeling induced by pressure overload and explore its potential mechanisms. Left ventricular (LV) KCP expression was measured with real-time quantitative PCR, western blotting, and immunofluorescence staining in pressure overload-induced cardiac remodeling in mice. Cardiac function and remodeling were evaluated in wide-type (WT) mice and KCP knockout (KO) mice by echocardiography, which were further confirmed by histological analysis with hematoxylin and eosin and Masson staining. RNA sequence was performed with LV tissue from WT and KO mice to identify differentially expressed genes and related signaling pathways. Primary cardiac fibroblasts (CFs) were used to validate the regulatory role and potential mechanisms of KCP during fibrosis. KCP was down-regulated in the progression of cardiac remodeling induced by pressure overload, and was mainly expressed in fibroblasts. KCP deficiency significantly aggravated pressure overload-induced cardiac dysfunction and remodeling. RNA sequence revealed that the role of KCP deficiency in cardiac remodeling was associated with cell division, cell cycle, and P53 signaling pathway, while cyclin B1 (CCNB1) was the most significantly up-regulated gene. Further investigation invivo and invitro suggested that KCP deficiency promoted the proliferation of CFs via P53/P21/CCNB1 pathway. Taken together, these results suggested that KCP deficiency aggravates cardiac dysfunction and remodeling induced by pressure overload via P53/P21/CCNB1 signaling in mice.

DEL-1 deficiency aggravates pressure overload-induced heart failure by promoting neutrophil infiltration and neutrophil extracellular traps formation

Recent studies have shown that neutrophils play an important role in the development and progression of heart failure. Developmental endothelial locus-1 (DEL-1) is an anti-inflammatory glycoprotein that has been found to have protective effects in various cardiovascular diseases. However, the role of DEL-1 in chronic heart failure is not well understood. In a mouse model of pressure overload-induced non-ischemic cardiac failure, we found that neutrophil infiltration in the heart increased and DEL-1 levels decreased in the early stages of heart failure. DEL-1 deficiency worsened pressure overload-induced cardiac dysfunction and remodeling in mice. Mechanistically, DEL-1 deficiency promotes neutrophil infiltration and the formation of neutrophil extracellular traps (NETs) through the regulation of P38 signaling. In vitro experiments showed that DEL-1 can inhibit P38 signaling and NETs formation in mouse neutrophils in a MAC-1-dependent manner. Depleting neutrophils, inhibiting NETs formation, and inhibiting P38 signaling all reduced the exacerbation of heart failure caused by DEL-1 deletion. Overall, our findings suggest that DEL-1 deficiency worsens pressure overload-induced heart failure by promoting neutrophil infiltration and NETs formation.

Modulation of cardiac cAMP signaling by AMPK and its adjustments in pressure overload-induced myocardial dysfunction in rat and mouse.

The beta-adrenergic system is a potent stimulus for enhancing cardiac output that may become deleterious when energy metabolism is compromised as in heart failure. We thus examined whether the AMP-activated protein kinase (AMPK) that is activated in response to energy depletion may control the beta-adrenergic pathway. We studied the cardiac response to beta-adrenergic stimulation of AMPKα2-/- mice or to pharmacological AMPK activation on contractile function, calcium current, cAMP content and expression of adenylyl cyclase 5 (AC5), a rate limiting step of the beta-adrenergic pathway. In AMPKα2-/- mice the expression of AC5 (+50%), the dose response curve of left ventricular developed pressure to isoprenaline (p<0.001) or the response to forskolin, an activator of AC (+25%), were significantly increased compared to WT heart. Similarly, the response of L-type calcium current to 3-isobutyl-l-methylxanthine (IBMX), a phosphodiesterase inhibitor was significantly higher in KO (+98%, p<0.01) than WT (+57%) isolated cardiomyocytes. Conversely, pharmacological activation of AMPK by 5-aminoimidazole-4-carboxamide riboside (AICAR) induced a 45% decrease in AC5 expression (p<0.001) and a 40% decrease of cAMP content (P<0.001) as measured by fluorescence resonance energy transfer (FRET) compared to unstimulated rat cardiomyocytes. Finally, in experimental pressure overload-induced cardiac dysfunction, AMPK activation was associated with a decreased expression of AC5 that was blunted in AMPKα2-/- mice. The results show that AMPK activation down-regulates AC5 expression and blunts the beta-adrenergic cascade. This crosstalk between AMPK and beta-adrenergic pathways may participate in a compensatory energy sparing mechanism in dysfunctional myocardium.

Effect of SH2B1 on glucose metabolism during pressure overload-induced cardiac hypertrophy and cardiac dysfunction.

This study mainly explored the effect and mechanism of Src homology 2 (SH2) B adaptor protein 1 (SH2B1) on cardiac glucose metabolism during pressure overload-induced cardiac hypertrophy and dysfunction. A pressure-overloaded cardiac hypertrophy model was constructed, and SH2B1-siRNA was injected through the tail vein. Haematoxylin and eosin (H&E) staining was used to detect myocardial morphology. ANP, BNP, β-MHC and the diameter of myocardial fibres were quantitatively measured to evaluate the degree of cardiac hypertrophy, respectively. GLUT1, GLUT4, and IR were detected to assess cardiac glucose metabolism. Cardiac function was determined by echocardiography. Then, glucose oxidation and uptake, glycolysis and fatty acid metabolism were assessed in Langendorff perfusion of hearts. Finally, PI3K/AKT activator was used to further explore the relevant mechanism. The results showed that during cardiac pressure overload, with the aggravation of cardiac hypertrophy and dysfunction, cardiac glucose metabolism and glycolysis increased, and fatty acid metabolism decreased. After SH2B1-siRNA transfection, cardiac SH2B1 expression was knocked down, and the degree of cardiac hypertrophy and dysfunction was alleviated compared with the Control-siRNA transfected group. Simultaneously, cardiac glucose metabolism and glycolysis were reduced, and fatty acid metabolism was enhanced. The SH2B1 expression knockdown mitigated the cardiac hypertrophy and dysfunction by reducing cardiac glucose metabolism. After using PI3K/AKT activator, the effect of SH2B1 expression knockdown on cardiac glucose metabolism was reversed during cardiac hypertrophy and dysfunction. Collectively, SH2B1 regulated cardiac glucose metabolism by activating the PI3K/AKT pathway during pressure overload-induced cardiac hypertrophy and cardiac dysfunction.

Semaphorin3A Exacerbates Cardiac Microvascular Rarefaction in Pressure Overload-Induced Heart Disease.

Microvascular endothelial cells (MiVECs) impair angiogenic potential, leading to microvascular rarefaction, which is a characteristic feature of chronic pressure overload-induced cardiac dysfunction. Semaphorin3A (Sema3A) is a secreted protein upregulated in MiVECs following angiotensin II (Ang II) activation and pressure overload stimuli. However, its role and mechanism in microvascular rarefaction remain elusive. The function and mechanism of action of Sema3A in pressure overload-induced microvascular rarefaction, is explored, through an Ang II-induced animal model of pressure overload. RNA sequencing, immunoblotting analysis, enzyme-linked immunosorbent assay, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and immunofluorescence staining results indicate that Sema3A is predominantly expressed and significantly upregulated in MiVECs under pressure overload. Immunoelectron microscopy and nano-flow cytometry analyses indicate small extracellular vesicles (sEVs), with surface-attached Sema3A, to be a novel tool for efficient release and delivery of Sema3A from the MiVECs to extracellular microenvironment. To investigate pressure overload-mediated cardiac microvascular rarefaction and cardiac fibrosis in vivo, endothelial-specific Sema3A knockdown mice are established. Mechanistically, serum response factor (transcription factor) promotes the production of Sema3A; Sema3A-positive sEVs compete with vascular endothelial growth factor A to bind to neuropilin-1. Therefore, MiVECs lose their ability to respond to angiogenesis. In conclusion, Sema3A is a key pathogenic mediator that impairs the angiogenic potential of MiVECs, which leads to cardiac microvascular rarefaction in pressure overload-induced heart disease.

Genetic deletion of CXCL10 prevents pressure overload-induced cardiac hypertrophy and dysfunction

Rationale: Heart failure is an inflammatory disease characterized by elevated serum levels of CXCL9 and CXCL10, which are CXCR3 receptor ligands. Both chemokines are expressed by cardiac fibroblasts and myeloid cells in mice subjected to pressure overload induced by transverse aortic constriction (TAC) surgery. Although it is known that CXCR3-/- animals are protected from cardiac remodeling induced by TAC, the role of CXCL10 is unknown. Objective: To assess the role of CXCL10 in the cardiac remodeling response to cardiac pressure overload induced by TAC in mice. Methods and results: We subjected wild-type and CXCL10-/- (male and female) mice to TAC surgery. After 6 weeks, we observed that the CXCL10-/- mice showed an attenuation of heart weight when compared to the increase observed in wild-type animals. At the cellular level, the genetic deletion of CXCL10 prevented the TAC-induced increase in the cross-sectional area of cardiac tissue. In addition, echocardiographic changes, including dilation and cardiac dysfunction, induced by pressure overload in wild animals were blunted in CXCL10-/- mice. Conclusion: CXCL10 determines cardiac hypertrophy and cardiac dysfunction induced by pressure overload in mice. We are currently unraveling the mechanism through which CXCL10 can accelerate the development of heart failure in response to cardiac pressure overload. T cells are likely mediating these effects. 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.

Interleukin-12 alpha deficiency attenuates pressure overload-induced cardiac inflammation, hypertrophy, dysfunction, and heart failure progression

IL-12α plays an important role in modulating inflammatory response, fibroblast proliferation and angiogenesis through modulating macrophage polarization or T cell function, but its effect on cardiorespiratory fitness is not clear. Here, we studied the effect of IL-12α on cardiac inflammation, hypertrophy, dysfunction, and lung remodeling in IL-12α gene knockout (KO) mice in response to chronic pressure-overload produced by transverse aortic constriction (TAC). Our results showed that IL-12α KO significantly ameliorated TAC-induced left ventricular (LV) failure, as evidenced by a smaller decrease of LV ejection fraction and fractional shortening. IL-12α KO also significantly attenuated TAC-induced increase of LV weight, left atrial weight, lung weight, right ventricular weight, and their ratios to body weight or tibial length. In addition, IL-12α KO significantly attenuated TAC-induced LV leukocyte infiltration, fibrosis, cardiomyocyte hypertrophy, and lung inflammation and remodeling (such as lung fibrosis and vessel muscularization). Moreover, IL-12α KO significantly attenuated TAC-induced activation of CD4+ T cells and CD8+ T cells in the lung. Furthermore, IL-12α KO significantly suppressed accumulation and activation of pulmonary macrophages and dendritic cells. Taken together, these findings indicate that inhibition of IL-12α is effective in attenuating pressure overload-induced cardiac inflammation, heart failure development, promoting transition from LV failure to lung remodeling and right ventricular hypertrophy. This work was supported by research grants R01HL161085, R01HL139797, P20GM104357, and P01HL51971 from NIH. 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.

Abstract 9582: Septin-4 Induces Cardiac Fibrosis After Pressure Overload

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.

Abstract P2022: Tipifarnib Mediated Protection By Reduction Of Circulating Exosomes In Pressure Overloaded Cardiac Hypertrophy

Hypertrophic cardiomyopathy (HCM) in response to pathophysiological stress is one of the leading causes of heart failure. A growing body of evidence emphasizes the crucial role of exosomes and their modulated content in aggravating cardiac damage due to their inherent intercellular cross-talk abilities during cardiac remodeling. However, the role of circulating exosomes in HCM for the trafficking of pathogenic factors and remodeling the cardiac microenvironment is yet unclear. We investigated the effect of systemic exosome inhibition during cardiac dysfunction in a transverse aortic constriction (TAC) model of heart failure using a recently identified exosome biogenesis inhibitor, Tipifarnib initially developed as Farnesyl transferase inhibitor. In this study, 10-week-old C57BL6J male mice were randomized into three groups i.e., Sham, TAC and Tipifarnib treated (10 mg/kg) TAC. The untreated TAC mice gradually developed hypertrophy and had reduced cardiac functions with a significant increase in heart weight/body weight ratio, cardiomyocyte size and upregulation of hypertrophy and fibrosis associated genes expression by 8 weeks. On the contrary, Tipifarnib treated TAC mice showed remarkably improved cardiac left ventricular functions, reduced cardiac hypertrophy and fibrosis. Notably, Nanosight analysis indicated significantly higher serum exosomes concentration in TAC mice which were substantially suppressed with Tipifarnib treatment. The molecular analysis of the heart tissue revealed Tipifarnib treated TAC mice had reduced expression of the proteins involved in exosome biogenesis in comparison to untreated TAC mice. To gain insight into the cargo of these circulating exosomes, we performed the serum cytokine array and serum exosomes miRNA sequencing in untreated and Tipifarnib treated TAC mice. There was a marked reduction in inflammatory cytokines in serum and differentially expressed exosomal miRNAs with Tipifarnib treatment in comparison to the untreated TAC mice. Overall, our studies suggest the promising potential of Tipifarnib that effectively protects against pressure overload-induced cardiac remodeling and dysfunction by suppressing exosome secretion and altering hypertrophic and fibrotic gene expression.

Activation of transient receptor potential vanilloid 4 is involved in pressure overload-induced cardiac hypertrophy.

Previous studies, including our own, have demonstrated that transient receptor potential vanilloid 4 (TRPV4) is expressed in hearts and implicated in cardiac remodeling and dysfunction. However, the effects of TRPV4 on pressure overload-induced cardiac hypertrophy remain unclear. In this study, we found that TRPV4 expression was significantly increased in mouse hypertrophic hearts, human failing hearts, and neurohormone-induced hypertrophic cardiomyocytes. Deletion of TRPV4 attenuated transverse aortic constriction (TAC)-induced cardiac hypertrophy, cardiac dysfunction, fibrosis, inflammation, and the activation of NFκB - NOD - like receptor pyrin domain-containing protein 3 (NLRP3) in mice. Furthermore, the TRPV4 antagonist GSK2193874 (GSK3874) inhibited cardiac remodeling and dysfunction induced by TAC. In vitro, pretreatment with GSK3874 reduced the neurohormone-induced cardiomyocyte hypertrophy and intracellular Ca2+ concentration elevation. The specific TRPV4 agonist GSK1016790A (GSK790A) triggered Ca2+ influx and evoked the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). But these effects were abolished by removing extracellular Ca2+ or GSK3874. More importantly, TAC or neurohormone stimulation-induced CaMKII phosphorylation was significantly blocked by TRPV4 inhibition. Finally, we show that CaMKII inhibition significantly prevented the phosphorylation of NFκB induced by GSK790A. Our results suggest that TRPV4 activation contributes to pressure overload-induced cardiac hypertrophy and dysfunction. This effect is associated with upregulated Ca2+/CaMKII mediated activation of NFκB-NLRP3. Thus, TRPV4 may represent a potential therapeutic drug target for cardiac hypertrophy and dysfunction after pressure overload.

A novel and important role of UFM1‐binding protein 1 (UFBP1) in the regulation of ER and cardiac homeostasis

Protein modifications by ubiquitin and ubiquitin‐like proteins are emerging as an important mechanism regulating heart function. UFM1 (ubiquitin‐fold modifier 1) is a novel ubiquitin‐like protein that modifies protein targets via UFM1‐specific conjugation enzymes. Dysregulation of UFM1 modification, termed ufmylation, has been linked to multiple human diseases but its importance in the heart remains unclear. We have previously reported that ufmylation is upregulated in compensated, hypertrophic mouse hearts but decreased in failing human hearts. Inhibition of ufmylation by targeted ablation of the E3 UFM1 ligase 1 (UFL1) in mouse hearts resulted in dilated cardiomyopathy during ageing and increased the propensity to heart failure in response to hemodynamic stress. However, how ufmylation exerts its cardioprotective effect remains unclear. Here, we report that UFBP1 (UFM1 binding protein 1), an ER‐resident ufmylation target, is an important downstream effector of ufmylation in the heart. While deletion of UFBP1 in mouse hearts has no impact on ufmylation, the knockout (KO) mice developed dilated cardiomyopathy at resting condition, as indicated by left ventricular wall thinning, chamber dilatation and significantly decreased cardiac contractility at 6 months of age. Moreover, loss of UFBP1 exacerbated pressure overload‐induced cardiac remodeling and dysfunction, recapitulating multiple aspects of the phenotypes of UFL1‐deficient hearts. Mechanistically, UFL1 controls the expression of UFBP1. Depletion of both UFL1 and UFBP1 in cultured cardiomyocytes aggravated ER stress‐induced cell injury. Furthermore, excess ER stress is implicated in UFBP1KO hearts and is aggravated with the progression of cardiomyopathy. Interestingly, ER stress inducers upregulated the expression of ufmylation pathway components at both transcript and protein levels in cultured cardiomyocytes and mouse hearts, indicating an intimate cross‐talk between ufmylation and ER stress. Collectively, our data identify a novel UFM1‐UFL1‐UFBP1 axis in constraining pathological cardiac remodeling and maintaining ER homeostasis.

Tumor Necrosis Factor-α-Induced Protein 8-Like 2 Ameliorates Cardiac Hypertrophy by Targeting TLR4 in Macrophages.

Background Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2), a novel immunoregulatory protein, has been reported to regulate inflammation and apoptosis. The role of TIPE2 in cardiovascular disease, especially cardiac hypertrophy, has not been elucidated. Thus, the aim of the present study was to explore the role of TIPE2 in cardiac hypertrophy. Methods Mice were subjected to aortic banding (AB) to induce an adverse hypertrophic model. To overexpress TIPE2, mice were injected with a lentiviral vector expressing TIPE2. Echocardiographic and hemodynamic analyses were used to evaluate cardiac function. Neonatal rat cardiomyocytes (NRCMs) and mouse peritoneal macrophages (MPMs) were isolated and stimulated with angiotensin II. NRCMs and MPM were also cocultured and stimulated with angiotensin II. Cells were transfected with Lenti-TIPE2 to overexpress TIPE2. Results TIPE2 expression levels were downregulated in hypertrophic mouse hearts and in macrophages in heart tissue. TIPE2 overexpression attenuated pressure overload-induced cardiac hypertrophy, fibrosis, and cardiac dysfunction. Moreover, we found that TIPE2 overexpression in neonatal cardiomyocytes did not relieve the angiotensin II-induced hypertrophic response in vitro. Furthermore, TIPE2 overexpression downregulated TLR4 and NF-κB signaling in macrophages but not in cardiomyocytes, which led to diminished inflammation in macrophages and consequently reduced the activation of hypertrophic Akt signaling in cardiomyocytes. TLR4 inhibition by TAK-242 did not enhance the antihypertrophic effect of TIPE2 overexpression. Conclusions The present study indicated that TIPE2 represses macrophage activation by targeting TLR4, subsequently inhibiting cardiac hypertrophy.