Cardiomyocyte Hypertrophy Research Articles

3D Imaging Reveals Complex Microvascular Remodeling in the Right Ventricle in Pulmonary Hypertension.

Pathogenic concepts of right ventricular (RV) failure in pulmonary arterial hypertension focus on a critical loss of microvasculature. However, the methods underpinning prior studies did not take into account the 3-dimensional (3D) aspects of cardiac tissue, making accurate quantification difficult. We applied deep-tissue imaging to the pressure-overloaded RV to uncover the 3D properties of the microvascular network and determine whether deficient microvascular adaptation contributes to RV failure. Heart sections measuring 250-µm-thick were obtained from mice after pulmonary artery banding (PAB) or debanding PAB surgery and properties of the RV microvascular network were assessed using 3D imaging and quantification. Human heart tissues harvested at the time of transplantation from pulmonary arterial hypertension cases were compared with tissues from control cases with normal RV function. Longitudinal 3D assessment of PAB mouse hearts uncovered complex microvascular remodeling characterized by tortuous, shorter, thicker, highly branched vessels, and overall preserved microvascular density. This remodeling process was reversible in debanding PAB mice in which the RV function recovers over time. The remodeled microvasculature tightly wrapped around the hypertrophied cardiomyocytes to maintain a stable contact surface to cardiomyocytes as an adaptation to RV pressure overload, even in end-stage RV failure. However, microvasculature-cardiomyocyte contact was impaired in areas with interstitial fibrosis where cardiomyocytes displayed signs of hypoxia. Similar to PAB animals, microvascular density in the RV was preserved in patients with end-stage pulmonary arterial hypertension, and microvascular architectural changes appeared to vary by etiology, with patients with pulmonary veno-occlusive disease displaying a lack of microvascular complexity with uniformly short segments. 3D deep tissue imaging of the failing RV in PAB mice, pulmonary hypertension rats, and patients with pulmonary arterial hypertension reveals complex microvascular changes to preserve the microvascular density and maintain a stable microvascular-cardiomyocyte contact. Our studies provide a novel framework to understand microvascular adaptation in the pressure-overloaded RV that focuses on cell-cell interaction and goes beyond the concept of capillary rarefaction.

RETROSPECTIVE ANALYSIS OF CLINICAL, EPIDEMIOLOGY AND PATHOMORPHOLOGICAL FEATURES OF MYOCARDIAL INFARCTION

The aim of this study is to conduct a retrospective analysis of the clinical, epidemiological, and pathomorphological features of myocardial infarction. Materials and methods: A retrospective analysis was conducted using data from medical records and autopsy results from the pathology department for the year 2023. Results. In 2023, the pathology department performed 182 autopsies, with 123 patients diagnosed with acute, repeated, or recurrent myocardial infarction (MI), accounting for 67.6% of all autopsies. The average age of the deceased was 74.4±0.9 years, with 81.3% being persons of incapacitated age. Women comprised the majority at 55.3% (T=2784.5; p=0.001). The highest proportion of MI-related deaths occurred in September, accounting for 12.2%. In the pathological diagnosis, myocardial infarction (MI) was the primary disease, with varying localization of the pathological process in the left ventricle. Transmural MI was detected in 95.9% of the deceased, while subendocardial MI was found in 4.1%. Acute MI developed against the background of a previously experienced MI in 43.9% of cases, and in 5.5% of cases, it was a repeated acute recurrent MI. Changes in the aorta, coronary vessels, and basal vessels of the brain were observed in 100.0% of the cases, with hypertensive disease detected in 95.9% of cases. Acute cardiopulmonary failure with the development of pulmonary edema was the immediate cause of death in 95.9% of cases. Macroscopic examination revealed hypertrophy of the left ventricle. The myocardium appeared reddish-brown and elastic, with white layers. Brown-gray infarct areas of different sizes were present in the left ventricular wall, while in cases of repeated acute myocardial infarction, the affected area was gray-white and hard to the touch. Microscopically, hypertrophy of cardiomyocytes of the left ventricle with perinuclear lipofuscinosis was observed in all deceased individuals. Sections of the infarct zone contained homogenized, anucleated, fragmented cardiomyocytes, dilated full blood vessels with small perivascular hemorrhages, and neutrophilic-macrophage infiltrates of varying intensity. Large centers of cardiosclerosis were detected in cases of repeated MI, while signs of angiogenesis with the formation of granulation tissue were observed in cases of recurrent MI. Conclusion. In 2023, 123 autopsies revealed a diagnosis of acute, repeated, or recurrent myocardial infarction (MI) in 67.6% of all cases. The average age of the deceased was 74.4±0.9 years, with 81.3% being individuals of incapacitated age. There was a predominance of women, accounting for 55.3% of cases. Acute MI developed against the background of a previously experienced one in 43.9% of the deceased, while repeated acute recurrent MI was detected in 5.5% of cases. Pathomorphological changes corresponded to the typical features of MI depending on its age.

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.

Identification of Prominin-2 as a new player of cardiomyocyte senescence in the aging heart.

The aging heart is characterized by a number of structural changes leading to ventricular stiffness, impaired resistance to stress and increased risk of developing heart failure (HF). Genetic or pharmacological removal of senescent cells has recently demonstrated the possibility to relieve some cardiac aging features such as hypertrophy and fibrosis. However, the contribution of the different cell types in cardiac aging remains fragmentary due to a lack of cell-specific markers. Cardiomyocytes undergo post-mitotic senescence in response to telomere damage, characterized by persistent DNA damage response and expression of the classical senescence markers p21 and p16, which are shared by many other cell types. In the present study, we used transcriptomic approaches to discover new markers specific for cardiomyocyte senescence. We identified Prominin2 (Prom2), encoding a transmembrane glycoprotein, as the most upregulated gene in cardiomyocytes of aged mice compared to young mice. We showed that Prom2 was upregulated by a p53-dependent pathway in stress-induced premature senescence. Prom2 expression correlated with cardiomyocyte hypertrophy in the hearts of aged mice and was increased in atrial samples of patients with HF with preserved ejection fraction. Consistently, Prom2 overexpression was sufficient to drive senescence, hypertrophy and resistance to cytotoxic stress while Prom2 shRNA silencing inhibited these features in doxorubicin-treated cardiac cells. In conclusion, we identified Prom2 as a new player of cardiac aging, linking cardiomyocyte hypertrophy to senescence. These results could provide a better understanding and targeting of cell-type specific senescence in age-associated cardiac diseases.

Localized translation and sarcomere maintenance requires ribosomal protein SA in mice.

Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using proximity labeling, we identified Ribosomal Protein SA (RPSA) as a Z-line protein. In cultured cardiomyocytes, the loss of RPSA led to impaired local protein translation and reduced sarcomere integrity. By employing CAS9 expressing mice along with adeno-associated viruses expressing CRE recombinase and single-guide RNAs targeting Rpsa, we knocked out Rpsa in vivo and observed mis-localization of ribosomes and diminished local translation. These genetic mosaic mice with Rpsa knockout in a subset of cardiomyocytes developed dilated cardiomyopathy, featuring atrophy of RPSA-deficient cardiomyocytes, compensatory hypertrophy of unaffected cardiomyocytes, left ventricular dilation, and impaired contractile function. We demonstrate that RPSA C-terminal domain is sufficient for localization to the Z-lines and that if the microtubule network is disrupted RPSA loses its sarcomeric localization. These findings highlight RPSA as a ribosomal factor essential for ribosome localization to the Z-line, facilitating local translation and sarcomere maintenance.

Morphological aspects of the heart of young rats subjected to high and medium intensity progressive resistance physical exercise protocols

Morphological aspects of the heart of young rats subjected to high and medium intensity progressive resistance physical exercise protocols

Proline metabolic reprogramming modulates cardiac remodeling induced by pressure overload in the heart.

Metabolic reprogramming is critical in the onset of pressure overload-induced cardiac remodeling. Our study reveals that proline dehydrogenase (PRODH), the key enzyme in proline metabolism, reprograms cardiomyocyte metabolism to protect against cardiac remodeling. We induced cardiac remodeling using transverse aortic constriction (TAC) in both cardiac-specific PRODH knockout and overexpression mice. Our results indicate that PRODH expression is suppressed after TAC. Cardiac-specific PRODH knockout mice exhibited worsened cardiac dysfunction, while mice with PRODH overexpression demonstrated a protective effect. In addition, we simulated cardiomyocyte hypertrophy in vitro using neonatal rat ventricular myocytes treated with phenylephrine. Through RNA sequencing, metabolomics, and metabolic flux analysis, we elucidated that PRODH overexpression in cardiomyocytes redirects proline catabolism to replenish tricarboxylic acid cycle intermediates, enhance energy production, and restore glutathione redox balance. Our findings suggest PRODH as a modulator of cardiac bioenergetics and redox homeostasis during cardiac remodeling induced by pressure overload. This highlights the potential of PRODH as a therapeutic target for cardiac remodeling.

Qiangxinyin formula protects against isoproterenol-induced cardiac hypertrophy

Heart failure is a life-threatening cardiovascular disease and characterized by cardiac hypertrophy, inflammation and fibrosis. The traditional Chinese medicine formula Qiangxinyin (QXY) is effective for the treatment of heart failure while the underlying mechanism is not clear. This study aims to identify the active ingredients of QXY and explore its mechanisms protecting against cardiac hypertrophy. We found that QXY significantly protected against isoproterenol (ISO)-induced cardiac hypertrophy and dysfunction in zebrafish. Eight compounds, including benzoylmesaconine (BMA), atractylenolide I (ATL I), icariin (ICA), quercitrin (QUE), psoralen (PRN), kaempferol (KMP), ferulic acid (FA) and protocatechuic acid (PCA) were identified from QXY. PRN, KMP and icaritin (ICT), an active pharmaceutical ingredient of ICA, prevented ISO-induced cardiac hypertrophy and dysfunction in zebrafish. In H9c2 cardiomyocyte treated with ISO, QXY significantly blocked the calcium influx, reduced intracellular lipid peroxidative product MDA, stimulated ATP production and increased mitochondrial membrane potential. QXY also inhibited ISO-induced cardiomyocyte hypertrophy and cytoskeleton reorganization. Mechanistically, QXY enhanced the phosphorylation of Smad family member 2 (SMAD2) and myosin phosphatase target subunit-1 (MYPT1), and suppressed the phosphorylation of myosin light chain (MLC). In conclusion, PRN, KMP and ICA are the main active ingredients of QXY that protect against ISO-induced cardiac hypertrophy and dysfunction largely via the blockage of calcium influx and inhibition of mitochondrial dysfunction as well as cytoskeleton reorganization.

Striatin plays a major role in angiotensin II-induced cardiomyocyte and cardiac hypertrophy in mice in vivo.

The three striatins (STRN, STRN3, STRN4) form the core of STRiatin-Interacting Phosphatase and Kinase (STRIPAK) complexes. These place protein phosphatase 2A (PP2A) in proximity to protein kinases thereby restraining kinase activity and regulating key cellular processes. Our aim was to establish if striatins play a significant role in cardiac remodelling associated with cardiac hypertrophy and heart failure. All striatins were expressed in control human hearts, with up-regulation of STRN and STRN3 in failing hearts. We used mice with global heterozygote gene deletion to assess the roles of STRN and STRN3 in cardiac remodelling induced by angiotensin II (AngII; 7 days). Using echocardiography, we detected no differences in baseline cardiac function or dimensions in STRN+/- or STRN3+/- male mice (8 weeks) compared with wild-type littermates. Heterozygous gene deletion did not affect cardiac function in mice treated with AngII, but the increase in left ventricle mass induced by AngII was inhibited in STRN+/- (but not STRN3+/-) mice. Histological staining indicated that cardiomyocyte hypertrophy was inhibited. To assess the role of STRN in cardiomyocytes, we converted the STRN knockout line for inducible cardiomyocyte-specific gene deletion. There was no effect of cardiomyocyte STRN knockout on cardiac function or dimensions, but the increase in left ventricle mass induced by AngII was inhibited. This resulted from inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. The data indicate that cardiomyocyte striatin is required for early remodelling of the heart by AngII and identify the striatin-based STRIPAK system as a signalling paradigm in the development of pathological cardiac hypertrophy.

Metformin induction of heat shock factor 1 activation and the mitochondrial unfolded protein response alleviate cardiac remodeling in spontaneously hypertensive rats.

Heart failure and cardiac remodeling are both characterized by mitochondrial dysfunction. Healthy mitochondria are required for adequate contractile activity and appropriate regulation of cell survival. In the mammalian heart, enhancement of the mitochondrial unfolded protein response (UPRmt) is cardioprotective under pressure overload conditions. We explored the UPRmt and the underlying regulatory mechanism in terms of hypertension-induced cardiac remodeling and the cardioprotective effect of metformin. Male spontaneously hypertensive rats and angiotensin II-treated neonatal rat cardiomyocytes were used to induce cardiac hypertrophy. The results showed that hypertension induced the formation of aberrant mitochondria, characterized by a reduced mtDNA/nDNA ratio and swelling, as well as lower levels of mitochondrial complexes I to V and inhibition of the expression of one protein subunit of each of complexes I to IV. Such changes eventually enlarged cardiomyocytes and increased cardiac fibrosis. Metformin treatment increased the mtDNA/nDNA ratio and regulated the UPRmt, as indicated by increased expression of activating transcription factor 5, Lon protease 1, and heat shock protein 60, and decreased expression of C/EBP homologous protein. Thus, metformin improved mitochondrial ultrastructure and function in spontaneously hypertensive rats. Invitro analyses revealed that metformin reduced the high levels of angiotensin II-induced mitochondrial reactive oxygen species in such animals and stimulated nuclear translocation of heat shock factor 1 (HSF1). Moreover, HSF1 small-interfering RNA reduced the metformin-mediated improvements in mitochondrial morphology and the UPRmt by suppressing hypertrophic signals and cardiomyocyte apoptosis. These results suggest that HSF1/UPRmt signaling contributes to the beneficial effects of metformin. Metformin-mediated targeting of mitochondrial protein homeostasis and modulation of HSF1 levels have potential therapeutic implications in terms of cardiac remodeling.

Triiodo-L-thyronine (T3) downregulates Npr1 gene (coding for natriuretic peptide receptor-A) transcription in H9c2 cells: involvement of β-AR-ROS signaling.

Natriuretic peptide receptor-A (NPR-A) signaling system is considered as an intrinsic productive mechanism of the heart that opposes abnormal cardiac remodeling and hypertrophic growth. NPR-A is coded by Npr1 gene, and its expression is downregulated in the hypertrophied heart. We sought to examine the levels of Npr1 gene transcription in triiodo-L-thyronine (T3) treated hypertrophied cardiomyocyte (H9c2) cells, in vitro, and also the involvement of β-adrenergic receptor (β-AR) - Reactive oxygen species (ROS) signaling system in the down-regulation of Npr1 transcription also studied. Anti-hypertrophic Npr1 gene transcription was monitored in control and T3-treated (dose and time dependent) H9c2 cells, using a real time PCR method. Further, cell size, intracellular cGMP, ROS, hypertrophy markers (ANP, BNP, α-sk, α-MHC and β-MHC), β-AR, and protein kinase cGMP-dependent 1 (PKG-I) genes expression were also determined. The intracellular cGMP and ROS levels were determined by ELISA and DCF dye method, respectively. In addition, to neutralize T3 mediated ROS generation, H9c2 cells were treated with T3 in the presence and absence of antioxidants [curcumin (CU) or N-acetyl-L-cysteine (NAC)]. A dose dependent (10 pM, 100 pM, 1 nM and 10 nM) and time dependent (12 h, 24 h and 48 h) down-regulation of Npr1 gene transcription (20, 39, 60, and 74% respectively; 18, 55, and 85%, respectively) were observed in T3-treated H9c2 cells as compared with control cells. Immunofluorescence analysis also revealed that a marked down regulation of NPR- A protein in T3-treated cells as compared with control cells. Further, a parallel downregulation of cGMP and PKG-I (2.4 fold) were noticed in the T3-treated cells. In contrast, a time dependent increased expression of β-AR (60, 72, and 80% respectively) and ROS (26, 48, and 74%, respectively) levels were noticed in T3-treated H9c2 cells as compared with control cells. Interestingly, antioxidants, CU or NAC co-treated T3 cells displayed a significant reduction in ROS (69 and 81%, respectively) generation and to increased Npr1 gene transcription (81 and 88%, respectively) as compared with T3 alone treated cells. Our result suggest that down regulation of Npr1 gene transcription is critically involved in T3- induced hypertrophic growth in H9c2 cells, and identifies the cross-talk between T3-β-AR-ROS and NPR-A signaling.

Exercise-induced changes in myocardial glucose utilization during periods of active cardiac growth

Exercise training can promote physiological cardiac growth, which has been suggested to involve changes in glucose metabolism to facilitate hypertrophy of cardiomyocytes. In this study, we used a dietary, in vivo isotope labeling approach to examine how exercise training influences the metabolic fate of carbon derived from dietary glucose in the heart during acute, active, and established phases of exercise-induced cardiac growth. Male and female FVB/NJ mice were subjected to treadmill running for up to 4 weeks and cardiac growth was assessed by gravimetry. Cardiac metabolic responses to exercise were assessed via in vivo tracing of [13C6]-glucose via mass spectrometry and nuclear magnetic resonance. We found that the half-maximal cardiac growth response was achieved by approximately 1 week of daily exercise training, with near maximal growth observed in male mice with 2 weeks of training; however, female mice were recalcitrant to exercise-induced cardiac growth and required a higher daily intensity of exercise training to achieve significant, albeit modest, increases in cardiac mass. We also found that increases in the energy charge of adenylate and guanylate nucleotide pools precede exercise-induced changes in cardiac size and were associated with higher glucose tracer enrichment in the TCA pool and in amino acids (aspartate, glutamate) sourced by TCA intermediates. Our data also indicate that the activity of collateral biosynthetic pathways of glucose metabolism may not be markedly altered by exercise. Overall, this study provides evidence that metabolic remodeling in the form of heightened energy charge and increased TCA cycle activity and cataplerosis precedes cardiac growth caused by exercise training in male mice.

RICH1 is a novel key suppressor of isoproterenol‑ or angiotensin II‑induced cardiomyocyte hypertrophy.

Cardiac hypertrophy is one of the key processes in the development of heart failure. Notably, small GTPases and GTPase‑activating proteins (GAPs) serve essential roles in cardiac hypertrophy. RhoGAP interacting with CIP4 homologs protein 1 (RICH1) is a RhoGAP that can regulate Cdc42/Rac1 and F‑actin dynamics. RICH1 is involved in cell proliferation and adhesion; however, to the best of our knowledge, its role in cardiac hypertrophy remains unknown. In the present study, the role of RICH1 in cardiomyocyte hypertrophy was assessed. Cell viability was analyzed using the Cell Counting Kit‑8 assay and cells surface area (CSA) was determined by cell fluorescence staining. Reverse transcription‑quantitative PCR and western blotting were used to assess the mRNA expression levels of hypertrophic marker genes, such as Nppa, Nppb and Myh7, and the protein expression levels of RICH1, respectively. RICH1 was shown to be downregulated in isoproterenol (ISO)‑ or angiotensin II (Ang II)‑treated H9c2 cells. Notably, overexpression of RICH1 attenuated the upregulation of hypertrophy‑related markers, such as Nppa, Nppb and Myh7, and the enlargement of CSA induced by ISO and Ang II. By contrast, the knockdown of RICH1 exacerbated these effects. These findings suggested that RICH1 may be a novel suppressor of ISO‑ or Ang II‑induced cardiomyocyte hypertrophy. The results of the present study will be beneficial to further studies assessing the role of RICH1 and its downstream molecules in inhibiting cardiac hypertrophy.

Impact of 3-O-caffeoylquinic acid (chlorogenic acid) and Postn protein regulation on cardiomyocyte hypertrophy: Experimental insights and potential therapeutic implications

Background: Cardiac hypertrophy is characterized by an enlarged heart muscle, often due to increased workload or chronic heart diseases. Postn protein (periostin) plays a significant role in cardiac hypertrophy by influencing cardiomyocyte proliferation, inflammatory response, and fibrosis. The TGF-β and NF-κB pathways exacerbate inflammatory responses and structural changes in the heart during cardiac hypertrophy. Chlorogenic acid, found in plants, has antioxidant and anti-inflammatory properties, making it a potential candidate for inhibiting cardiac hypertrophy. Method: We used angiotensin II-induced H9c2 cardiomyocytes to construct a cell model of cardiac hypertrophy. Cells were categorized into control, model, shPostn, chlorogenic acid, and shPostn+chlorogenic acid groups. Various parameters, including cell surface area, activity, apoptosis rate, and expression levels of Postn, TGF-β/NF-κB pathway-related proteins, and mRNA, were evaluated. Results: Compared to the control group, the model group exhibited increased cell surface area and apoptosis rate, along with decreased cell activity. However, intervention with shPostn or chlorogenic acid led to significant reductions in cell surface area and apoptosis rate, coupled with increased cell viability. The shPostn+chlorogenic acid group showed further improvements. In addition, protein and mRNA expressions related to Postn, TGF-β/NF-κB pathways, and cardiac hypertrophy markers were upregulated in the model group but downregulated in the intervention groups, particularly the shPostn+chlorogenic acid group. Conclusion: Chlorogenic acid exerts its inhibitory effect on cardiac hypertrophy by modulating the TGF-β/NF-κB pathway through Postn protein. This study sheds light on potential therapeutic strategies for mitigating cardiac hypertrophy induced by angiotensin II and highlights chlorogenic acid as a promising candidate for further exploration in treating this condition.

CD8 Cell PHD2 Deficiency Amplifies Pressure Overload-Induced Cardiac Inflammation through Metabolic Reprogramming of CD8 Cells

Prolyl hydroxylase domain (PHD) proteins function as oxygen sensors and regulate various cellular metabolism. PHD2 is well-known for its role in oxygen sensing and has been implicated in cardiac hypertrophy and fibrosis through HIF-2α. However, the specific impact and the mechanism of CD8 cell PHD2 deficiency on cardiac inflammation and heart failure (HF) development remains unclear. Here, we explored the role of CD8 cell-specific PHD2 deficiency (CD8-PHD2 KO) in HF development by using transverse aortic constriction (TAC) mouse model. Our findings reveal that CD8 PHD2 knockout exacerbated TAC-induced left ventricular (LV) hypertrophy, as evidenced by increased LV weight relative to bodyweight or tibial length. CD8-PHD2 KO also leads to a significant increase in lung and right ventricular weight relative to bodyweight or tibial length following TAC. Furthermore, CD8-PHD2 KO worsens TAC-induced reductions in LV ejection fraction, LV fractional shortening, and LV dilatation. Additionally, CD8-PHD2 KO exacerbates TAC-induced LV cardiomyocyte hypertrophy, fibrosis, and immune cell infiltration. Moreover, our study demonstrated that CD8-PHD2 KO amplified the infiltration and activation of antigen-presenting cells, CD4 T cells, and CD8 T cells in the heart and lungs after TAC. At the molecular level, we characterized the PHD2-deficient CD8 cells showed typical features of anaerobic glycolysis, which were paralleled by augmented hypoxia-inducible factor 1α (HIF-1α) protein levels. Collectively, these results highlight the critical role of PHD2 in CD8 cells in regulating cardiac inflammation and HF development in response to systolic overload. National Institutes of Health Project Number 5R01HL161085-02. 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.

γδ T Cells Mediate Pressure Overload-Induced Cardiac Inflammation, Hypertrophy, Heart Failure Development, and Heart Failure Progression

Gamma delta T cells (γδ T cells) are T cells that have a γδ T-cell receptor (TCR) on their surface. γδ T cells display broad functional plasticity following recognition of infected or injured cells by production of cytokines and chemokines, and cytolysis of the infected or injured cells. However, the role of γδ T cells in heart failure (HF) development and HF progression is unknown. To investigate the role of γδ T cells in HF development and progression, we studied the effect of transverse aortic constriction (TAC) on cardiac inflammation, hypertrophy, and dysfunction in both wild type and TCRδ gene deficient (TCRδ−/−) mice, as TCRδ−/− mice have no γδ T cells. Interestingly, TCRδ−/− significantly attenuated TAC-induced left ventricular (LV) hypertrophy as evidenced by less increase of LV weight and its ratio to bodyweight or tibial length. TCRδ−/− also significantly attenuated TAC-induced increase of lung weight, right ventricular weight, and their ratio to bodyweight or tibial length. TCRδ−/− also significantly reduced TAC-induced decease of LV ejection fraction and LV fractional shortening, and LV dilatation. Furthermore, TCRδ−/− significantly attenuated TAC-induced LV cardiomyocyte hypertrophy, fibrosis, and immune cell infiltration. Moreover, we found that TCRδ−/− significantly reduced TAC-induced infiltration and activation of pulmonary antigen presenting cells, CD4 T cells and CD8 T cells. Together, these data indicate that γδ T cells exert an important role in promoting systolic overload-induced cardiac inflammation, HF development, and HF progression. This work was supported by NIH research grants R01HL161085, R01HL139797, P20GM104357, and P30GM149404. 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.

Internalized β2-Adrenergic Receptors Inhibit Subcellular Phospholipase C-Dependent Cardiac Hypertrophic Signaling

Chronic overstimulation of Gs-coupled b-adrenergic receptor (b-AR) signaling by catecholamines, induces pathological cardiac hypertrophy and ultimately heart failure. b1-ARs and b2-ARs are the two major subtypes of b-ARs present in the human heart and mediate the activity of sympathetic nervous system. It is known that b1-ARs and b2-ARs elicit significantly different or even opposite effects on cardiac functions such as contractility and heart failure, though they share overall sequence and structural homology, and both stimulate cAMP production. The molecular mechanisms underlying these distinct biological effects have not been fully elucidated. Previously, our laboratory demonstrated that a cAMP/EPAC/phospholipase Ce (PLCe)-mediated signaling pathway at the Golgi apparatus is important for regulation of cardiac hypertrophy. We also showed that this pathway is selectively stimulated by activation of b1-ARs localized at Golgi apparatus but not those at the plasma membrane (PM). Access to intracellular b1-ARs by catecholamines was mediated by the organic cation transporters (OCTs), and blockade of OCT3 inhibited catecholamine stimulated cardiomyocyte hypertrophy. In my current study using adult ventricular cardiac myocytes, I found that the activation of endosomal b2-ARs opposes stimulation of the prohypertrophic EPAC/PLCe pathway at the Golgi apparatus by either Angiotensin II (ATII), or the activation of Golgi localized b1-ARs by Dobutamine (Dob). b2-AR-dependent inhibition is at the level of PLCe since activation of b2-AR with salmeterol (Sal) blocks direct activation of EPAC/PLCe by the EPAC selective cAMP analog 8-(4-chlorophenylthio)-2′- O-methyl-cAMP (CPTOMe)-AM, and blocks PLCe activation by ATII which activates PLCe through a pathway that does not rely on EPAC. Blockade of internalization of b2-ARs with Dyngo-4A abolished this inhibitory signaling. Downstream of b2-ARs, I found that b2-AR-dependent PLCe inhibition is not mediated by PKA. Rather, blockade of Gi with PTX, or Gbg with gallein, inhibited the ability of b2-ARs to block Golgi PLCe activity. Additionally, inhibition of ERK activation by PD0325901 abolished b2-AR-mediated inhibition of Golgi PLCe activity. This supports a model where b2ARs internalized from the plasma membrane activate Gi releasing Gbg subunits leading to ERK activation and inhibition of PI hydrolysis at the Golgi apparatus, thereby inhibiting hypertrophic signaling by PLCe. In addition, we also showed PKD and HDAC phosphorylation downstream of PLCe signaling are significantly attenuated by b2ARs activation in mouse hearts. Consistent with the results from mice, b2ARs activation by Sal blunted cellular hypertrophy induced by b1-ARs activation by Dob in vitro measured by cell area and the expression of the hypertrophic marker, atrial natriuretic factor (ANF). Importantly, inhibition endosomal Gbg using a highly selective Gbg inhibitor (GRK2CT) fused to an endosomal targeting sequence (FYVE domain) prevent Sal mediated protective signaling in hypertrophy measurements in vitro. This indicates the inhibitory signaling downstream of b2ARs is indeed driven by endosomal Gbg. This study reveals a novel potential mechanism for b2-AR antagonism of the Epac/PLCe pathway that may contribute to the known protective effects of b2AR signaling on the development of heart failure. Elucidation of a mechanism for the anti-hypertrophic versus hypertrophic signaling balance from b1-ARs and b2-ARs gives critical insights into the development of new strategies for treatment of heart failure by targeting bAR subtypes. This work is supported by grant R35 GM127303-01 (A.V.S.). Preprint: https://www.biorxiv.org/content/10.1101/2023.06.07.544153v1. 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.

Genetic Deletion of Carboxypeptidase N Attenuates Angiotensin II-Induced Hypertension and Target Organ Damage

Objectives: Kininase I transforms bradykinin and kallidin, into kinin B1 receptor (B1R) agonists des-Arg9-BK and des-Arg10-kallidin, respectively. Carboxypeptidase M (CPM) is the membrane bound isoform of kininase I and carboxypeptidase N (CPN) is the isoform located in the plasma. Activation of the B1R has been implicated in the development of hypertension, diabetes and atherosclerosis. Pharmacologic inhibition of kininase I is associated with reductions in B1R expression, inflammation, oxidative stress, and improvements in insulin resistance. B1R can interact with aminopeptidase N (CD13) or high-mobility group box 1 (HMGB1) to mediate an inflammatory response. However, the exact role of kininase I in the development of hypertension and the regulation of these B1R-mediated signaling mechanisms remains unknown. In this study, we hypothesize that gene deletion of CPN will prevent B1R activation, reduce inflammation via interfering with CD13 and HMGB1 signaling, and ultimately attenuate the progression of hypertension and target organ damage. Methods and Results: To test our hypothesis we utilized 12-week-old male and female C57BL/6NJ wild-type (WT) and CPN gene knockout (CPNKO) mice. These mice were infused angiotensin II (Ang II, 600 ng/kg/min) or saline (vehicle) via osmotic minipumps for 2 weeks. Vehicle treated CPNKO and WT mice showed no significant difference in mean arterial blood pressure (MAP) (104 ±7 vs 102 ±5 mmHg, n=5, p≤0.05). Ang II-induced elevations in MAP were significantly reduced in CPNKO mice compared to WT mice infused with Ang II (129 ±8 vs 150 ±6 mmHg, n=5, p≤0.05). Interestingly, we did not find any significant sex differences in MAP in CPNKO groups. Additionally, Ang II treatment significantly increased cardiomyocyte hypertrophy in male and female WT mice, but this effect was blunted in CPNKO mice (n=5, p≤0.05; wheat germ agglutinin staining). Ang II infusion promoted collagen deposition in the aorta of WT male and female mice, but not in CPNKO mice (n=5, p≤0.05; picrosirius red/Masson’s trichrome). In male WT mice, Ang II significantly increased the immunoreactivity of CPM in the heart, but this was abrogated in CPNKO mice infused with Ang II (n=4, p≤0.05). Similarly, B1R was also upregulated in male WT mice hearts when compared to CPNKO mice after Ang II infusions (n=3, p≤0.05). We also found that Ang II significantly increased HMGB1 and CD13 in male WT hearts and brains, but not in CPNKO mice (n=4, p≤0.05). In addition, oxidative stress levels in the brain were significantly increased by Ang II in WT mice and was attenuated in CPNKO mice (n=4, p≤0.05; dihydroethidium staining). Conclusions: Genetic deletion of CPN attenuates Ang II-induced hypertension by reducing B1R mediated reductions in CD13 and HMGB1-mediated inflammatory responses and oxidative stress. Our data suggests that targeting kininase I may be a novel therapeutic target for hypertension and target organ damage. Funding: This study was supported by NHLBI/NIH 5R01HL153115 (S. Sriramula). 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.

Application of Digital Holographic Imaging to Monitor Real-Time Cardiomyocyte Hypertrophy Dynamics in Response to Norepinephrine Stimulation.

Cardiomyocyte hypertrophy, characterized by an increase in cell size, is associated with various cardiovascular diseases driven by factors including hypertension, myocardial infarction, and valve dysfunction. In vitro primary cardiomyocyte culture models have yielded numerous insights into the intrinsic and extrinsic mechanisms driving hypertrophic growth. However, due to limitations in current approaches, the dynamics of cardiomyocyte hypertrophic responses remain poorly characterized. In this study, we evaluate the application of the Holomonitor M4 digital holographic imaging microscope to track dynamic changes in cardiomyocyte surface area and volume in response to norepinephrine treatment, a model hypertrophic stimulus. The Holomonitor M4 permits non-invasive, label-free imaging of three-dimensional changes in cell morphology with minimal phototoxicity, thus enabling long-term imaging studies. Untreated and norepinephrine-stimulated primary neonatal rat cardiomyocytes were live-imaged on the Holomonitor M4, which was followed by image segmentation and single-cell tracking using the HOLOMONITOR App Suite software version 4.0.1.546. The 24 h treatment of cultured cardiomyocytes with norepinephrine increased cardiomyocyte spreading and optical volume as expected, validating the reliability of the approach. Single-cell tracking of both cardiomyocyte surface area and three-dimensional optical volume revealed dynamic increases in these parameters throughout the 24 h imaging period, demonstrating the potential of this technology to explore cardiomyocyte hypertrophic responses with greater temporal resolution; however, technological limitations were also observed and should be considered in the experimental design and interpretation of results. Overall, leveraging the unique advantages of the Holomonitor M4 digital holographic imaging system has the potential to empower future work towards understanding the molecular and cellular mechanisms underlying cardiomyocyte hypertrophy with enhanced temporal clarity.

The potential roles of exosomes in pathological cardiomyocyte hypertrophy mechanisms and therapy: A review.

Pathological cardiac hypertrophy, characterized by the enlargement of cardiac muscle cells, leads to serious cardiac conditions and stands as a major global health issue. Exosomes, comprising small lipid bilayer vesicles, are produced by various cell types and found in numerous bodily fluids. They play a pivotal role in intercellular communication by transferring bioactive cargos to recipient cells or activating signaling pathways in target cells. Exosomes from cardiomyocytes, endothelial cells, fibroblasts, and stem cells are key in regulating processes like cardiac hypertrophy, cardiomyocyte survival, apoptosis, fibrosis, and angiogenesis within the context of cardiovascular diseases. This review delves into exosomes' roles in pathological cardiac hypertrophy, first elucidating their impact on cell communication and signaling pathways. It then advances to discuss how exosomes affect key hypertrophic processes, including metabolism, fibrosis, oxidative stress, and angiogenesis. The review culminates by evaluating the potential of exosomes as biomarkers and their significance in targeted therapeutic strategies, thus emphasizing their critical role in the pathophysiology and management of cardiac hypertrophy.