Elizabeth J. Cartwright

Background: Heart failure (HF) is characterized by progressive adverse remodeling. However, the underlying molecular mechanisms governing the progression of pathological remodeling and ventricular dysfunction remain incompletely defined. Fibroblast growth factor receptor 1 (FGFR1) regulates growth factor signaling in the heart, yet its role in HF is limited. Methods: Proteomic and transcriptomic analyses were performed on left ventricular tissue from patients with HF to identify dysregulated remodeling pathways. FGFR1 expression and protein kinase B (AKT) signaling were examined in human hearts, in mice subjected to chronic isoprenaline infusion, and in cardiomyocyte-specific FGFR1 knockout (FGFR1^cKO) mice. In vitro studies were conducted in neonatal rat cardiomyocytes (NRCMs) and rat myoblasts (H9C2) using siRNA-mediated FGFR1 knockdown and stimulating AKT phosphorylation by the specific activator SC-79 under isoprenaline-induced β-adrenergic stress. Genetic restoration of FGFR1 was achieved using adeno-associated viral subtype 9 (AAV9) delivery of FGFR1 after the onset of remodeling, followed by echocardiographic, histological, and molecular analyses. Results: Human HF hearts exhibited broad dysregulation of remodeling pathways. Notably, AKT signaling was markedly suppressed. Reduced FGFR1 expression was accompanied by decreased AKT phosphorylation in human HF hearts. Consistently, chronic isoprenaline stress reduced cardiac FGFR1 expression in vivo and in vitro. Cardiomyocyte-specific FGFR1 deletion exacerbated systolic dysfunction and promoted ventricular dilation. Mechanistically, FGFR1 deficiency suppressed AKT activation, which contributed to cardiac pathological remodeling upon isoprenaline stress. In cardiomyocytes with FGFR1 knockdown, cardiomyocyte injury and maladaptive transcriptional responses were amplified under β-adrenergic stress. Of note, these detrimental effects were rescued by pharmacological activation of AKT using its activator, SC-79. Therapeutically, genetic FGFR1 re-establishment attenuated cardiac pathological remodeling and restored systolic function under longer-term isoprenaline stress, at least partially through maintenance of AKT activation. Conclusions: The current study has demonstrated that cardiac FGFR1 signaling exhibits cardioprotective potential that mitigates adverse ventricular remodeling and rescues cardiac function under pathological stress. Therefore, targeting FGFR1 signaling represents a potential strategy to prevent pathological remodeling and HF.

BACKGROUND: Endothelial defects in the heart are a contributor to cardiac pathological remodeling and dysfunction, which can be influenced by cardiomyocytes. Here, we explore cardioprotective crosstalk between cardiomyocytes and endothelial cells under diabetic conditions. METHODS: Type 2 diabetes was induced in male and female mice using a high-fat, high-sucrose diet in conjunction with low-dose streptozotocin. Cardiomyocyte-specific deletion of FGFR1 (fibroblast growth factor receptor 1) or KLB (beta-Klotho) was achieved by crossing floxed mice with αMHC (alpha-myosin heavy chain)-Cre transgenic mice, while the treatment potential was assessed by recombinant adeno-associated virus 9 delivery systems or administration of recombinant protein. Various in vitro models were used to investigate the mechanisms under a diabetes-like condition. The molecular mechanisms were explored through transcriptomics, proteomics, cytokine arrays, angiogenesis assays, histology, and luciferase reporter assays. RESULTS: Human diabetic hearts exhibited impaired angiogenic pathways with a marked reduction in myocardial capillary density. Multiomics profiling also revealed alterations in the FGFR1 (fibroblast growth factor receptor 1) pathway in mouse diabetic hearts. Both cardiomyocyte-specific FGFR1- and KLB- knockout mice exhibited reduced capillary abundance and developed decompensated cardiac remodeling. Mechanistic studies identified CEBPβ (CCAAT/enhancer binding protein beta) as a downstream transcription factor of the FGFR1/KLB pathway regulating MFG-E8 (milk fat globule-EGF factor 8) expression in cardiomyocytes. Cardiomyocyte-secreted MFG-E8 enhanced endothelial viability and branching. Importantly, the detrimental effects on the heart were rescued by corestoration of cardiac FGFR1 and KLB, whereas neither alone was sufficient, underscoring their synergistic action against diabetic stress. Finally, genetic or pharmacological enhancement of MFG-E8 reversed myocardial capillary rarefaction and led to an improvement in cardiac function, albeit with more pronounced therapeutic effects in males. CONCLUSIONS: Our findings demonstrate FGFR1/KLB-mediated cardioprotective mechanisms through cardiomyocyte-endothelial cell crosstalk and provide evidence that preserving adaptive remodeling represents a promising strategy to alleviate heart failure in both sexes.

Dysferlin is a membrane-associated protein that supports skeletal muscle function such that mutations in the DYSF gene can cause muscular dystrophy. Growing evidence suggests dysferlin regulates cardiac function, but this is less well understood. Tight regulation of the cardiac transverse-(T)-tubule network and excitation-contraction (EC) coupling mechanism is essential for healthy cardiac physiology. Remodelling of T-tubules and the EC coupling mechanism is observed during periods of cardiac stress and pathologies, promoting arrhythmias. However, little is known about how these mechanisms are regulated. Using a global dysferlin knockout (KO) mouse we have shown that the loss of dysferlin leads to a decrease in T-tubule density and the amplitude and rate of decay of the systolic Ca2+ transient. Further, electrical mapping of ex vivo DYSF KO hearts shows they are more susceptible to ventricular arrhythmias during isoprenaline induced stress. To induce stress in cardiac myocytes, we used hypo-osmotic shock injury (OSI) to damage T-tubule networks in cardiac myocytes, in vitro. As such, OSI increased T-tubule fragmentation and caused dysregulation of intracellular Ca2+ handling in dysferlin KO cells relative to WT controls. In summary, these findings demonstrate an essential role for dysferlin in cardiac physiology, especially during conditions of stress.

Increased consumption of ultra-processed foods (UPFs) is a risk factor for metabolic disorder–associated heart failure (HF). Here, we demonstrate that UPF-induced calpain-1 aggravated oxidative stress, thereby increasing high mobility group box 1–mediated (HMGB1-mediated) myocardial inflammation, which contributes to cardiac dysfunction. After illustrating the dysregulated inflammatory pathways in human and murine hearts upon metabolic stress, we revealed an increase in calpain-1 alongside profound oxidative stress and inflammation in the failing myocardium. Mechanistically, in neonatal rat cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes, HMGB1 was upregulated by calpain-1 and reactive oxygen species (ROS) upon stress of saturated and trans fatty acids. Consequently, HMGB1 promoted a proinflammatory response in macrophages. In contrast, inhibition of calpain or ROS efficiently repressed HMGB1 in cardiomyocytes. Therapeutically, either recombinant adeno-associated virus 9–delivered inhibitor of calpain-1 or its pharmacological inhibitor attenuated ROS and HMGB1-induced inflammation in the myocardium and mitigated HF in both male and female mice fed with an ultra-processed diet. Collectively, we have demonstrated the effects of suppressing calpain-1 and oxidative stress on alleviating myocardial inflammation via blockage of HMGB1 and cardiac dysfunction. The results provide a promising therapeutic strategy for preventing or treating HF in metabolic disorders.

BACKGROUND: Morbidity and mortality of heart failure with preserved ejection fraction (HFpEF) is increased in metabolic disorders. However, options for preventing and treating these prevalent outcomes are limited. Intramyocardial lipotoxicity contributes to cardiac dysfunction. Here, we investigate the mechanisms underlying EDEM2 (endoplasmic reticulum degradation–enhancing alpha-mannosidase–like protein 2) regulation of cardiac lipid homeostasis and assess strategies that inhibit the incidence and progression of HFpEF. METHODS: Metabolic stress was induced in C57BL/6 male mice using a high-fat diet and N ω-nitro-L-arginine methyl ester. The recombinant adeno-associated virus 9 delivery system was used for loss- and gain-of-function studies. Palmitic acid and oleic acid stimulation of rat cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes imitated a condition of high lipids in vitro. Molecular mechanisms were investigated via RNA sequencing, mass spectrometry proteomics, lipidomic analyses, transmission electron microscopy, histology, and luciferase reporter assays. RESULTS: In the human heart, we first detected lipid overload accompanied by a reduction of XBP1 (X-box binding protein 1) under metabolic stress. Thereafter, a decrease in EDEM2 was confirmed in human and mouse HFpEF hearts. Given that XBP1s (spliced X-box binding protein 1) is a transcription factor, EDEM2 was identified as its new target in cardiomyocytes. EDEM2 knockdown mice manifested lipid droplet accumulation and higher levels of triglycerides and diglycerides in the myocardium, aggravating oxidative stress, hypertrophy, and the onset and progression of HFpEF under metabolic stress. XBP1s ablation mice displayed a similar myocardial lipid disturbance and cardiac phenotypes, which were reversed by EDEM2 overexpression. Mechanistically, the findings obtained from rat cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes demonstrated that, in the presence of EDEM2, SEC23A mediated intracellular translocation of ATGL (adipose triglyceride lipase) under fatty acid stimulation, inhibiting ATGL degradation and excessive intracellular lipid droplets. Furthermore, the functional studies supported that EDEM2 prevention of lipid overload occurred in an ATGL-dependent manner. Therapeutically, cardiac XBP1s or EDEM2 restoration mitigated lipid deposition and preserved lipid profiles in the myocardium, thus preventing the development of HFpEF. CONCLUSIONS: We demonstrate a cardioprotective mechanism regulating myocardial lipid homeostasis. The findings provide a promising therapeutic target to prevent and treat HFpEF, a condition with limited treatment options.

Background: Heart failure with preserved ejection fraction (HFpEF) is linked to prolonged endoplasmic reticulum (ER) stress. P21‐activated kinase 2 (Pak2) facilitates a protective ER stress response. This study explores the mechanism and role of Pak2 in HFpEF pathology. Methods and Results: The HFpEF mouse model was established using a high‐fat diet combined with the nitric oxide synthase inhibitor Nω‐Nitro‐ l ‐arginine methyl ester (high‐fat diet+Nω‐Nitro‐ l ‐arginine methyl ester). The model exhibited the typical characteristics of HFpEF (cardiac hypertrophy, diastolic dysfunction with preserved systolic function, and lung edema) as determined by echocardiography and hemodynamic analysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling and dihydroethidium staining results showed that cell death and reactive oxygen species generation were higher in the high‐fat diet+Nω‐Nitro‐ l ‐arginine methyl ester–treated group. Transmission electron microscopy revealed disruption of the ER subcellular structures in the HFpEF mouse model, while western blot analysis confirmed reduced Pak2 phosphorylation and impaired inositol‐requiring enzyme 1/X‐box binding protein 1 splicing ER stress response signaling. Furthermore, H9c2 cells subjected to the palmitic acid–mediated metabolic stress developed temporal changes in unfolded protein response proteins and Pak2 activity. The inositol requiring enzyme 1/X‐box binding protein 1 splicing branch of unfolded protein response was impaired earlier than other branches. Overexpression of Pak2 by adenovirus in H9c2 cells sustained the activation of inositol requiring enzyme 1/X‐box binding protein 1 splicing. Pak2 deficiency in the mouse heart accelerated the HFpEF progression, and this phenomenon occurred as early as 10 weeks in Pak2 cardiomyocyte‐specific knockout mice. Conversely, adeno‐associated virus serotype 9–mediated Pak2 overexpression mitigated HFpEF symptoms, underscoring its protective role against HFpEF progression. Conclusions: Pak2 prevents HFpEF progression, exerting cardioprotective effects against ER stress. These insights underscore the therapeutic value of Pak2 in HFpEF.

Dysferlin is a 237 kDa membrane‐associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca²⁺ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)‐tubule network and the tight coordination of excitation–contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t‐tubules, and it is suspected that dysferlin regulates t‐tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction. image

Background: Prolonged activation of angiotensin II is the main mediator that contributes to the development of heart diseases, so converting angiotensin II into angiotensin 1‐7 has emerged as a new strategy to attenuate detrimental effects of angiotensin II. Prolylcarboxypeptidase is a lysosomal pro‐X carboxypeptidase that is able to cleave angiotensin II at a preferential acidic pH optimum. However, insufficient attention has been given to the cardioprotective functions of prolylcarboxylpeptidase. Methods and Results: We established a CRISPR/CRISPR‐associated protein 9–mediated global prolylcarboxylpeptidase‐knockout and adeno‐associated virus serotype 9–mediated cardiac prolylcarboxylpeptidase overexpression mouse models, which were challenged with the angiotensin II infusion (2 mg/kg per day) for 4 weeks, aiming to investigate the cardioprotective effect of prolylcarboxylpeptidase against hypertensive cardiac hypertrophy. Prolylcarboxylpeptidase expression was upregulated after 2 weeks of angiotensin II infusion and then became downregulated afterward in wild‐type mouse myocardium, suggesting its compensatory function against angiotensin II stress. Moreover, angiotensin II–treated prolylcarboxylpeptidase‐knockout mice showed aggravated cardiac remodeling and dampened cardiac contractility independent of hypertension. We also found that prolylcarboxylpeptidase localizes in cardiomyocyte lysosomes, and loss of prolylcarboxylpeptidase led to excessive angiotensin II levels in myocardial tissue. Further screening demonstrated that hypertrophic prolylcarboxylpeptidase‐knockout hearts showed upregulated extracellular signal‐regulated kinases 1/2 and downregulated protein kinase B activities. Importantly, adeno‐associated virus serotype 9–mediated restoration of prolylcarboxylpeptidase expression in prolylcarboxylpeptidase‐knockout hearts alleviated angiotensin II–induced hypertrophy, fibrosis, and cell death. Interestingly, the combination of adeno‐associated virus serotype 9–mediated prolylcarboxylpeptidase overexpression and an antihypertensive drug, losartan, likely conferred more effective protection than a single treatment protocol to mitigate angiotensin II–induced cardiac dysfunction. Conclusions: Our data demonstrate that prolylcarboxylpeptidase protects the heart from angiotensin II–induced hypertrophic remodeling by controlling myocardial angiotensin II levels.

Reduction in cardiac contractility is common in severe sepsis. However, the pathological mechanism is still not fully understood. Recently it has been found that circulating histones released after extensive immune cell death play important roles in multiple organ injury and disfunction, particularly in cardiomyocyte injury and contractility reduction. How extracellular histones cause cardiac contractility depression is still not fully clear. In this work, using cultured cardiomyocytes and a histone infusion mouse model, we demonstrate that clinically relevant histone concentrations cause significant increases in intracellular calcium concentrations with subsequent activation and enriched localization of calcium-dependent protein kinase C (PKC) α and βII into the myofilament fraction of cardiomyocytes in vitro and in vivo. Furthermore, histones induced dose-dependent phosphorylation of cardiac troponin I (cTnI) at the PKC-regulated phosphorylation residues (S43 and T144) in cultured cardiomyocytes, which was also confirmed in murine cardiomyocytes following intravenous histone injection. Specific inhibitors against PKCα and PKCβII revealed that histone-induced cTnI phosphorylation was mainly mediated by PKCα activation, but not PKCβII. Blocking PKCα also significantly abrogated histone-induced deterioration in peak shortening, duration and the velocity of shortening, and re-lengthening of cardiomyocyte contractility. These in vitro and in vivo findings collectively indicate a potential mechanism of histone-induced cardiomyocyte dysfunction driven by PKCα activation with subsequent enhanced phosphorylation of cTnI. These findings also indicate a potential mechanism of clinical cardiac dysfunction in sepsis and other critical illnesses with high levels of circulating histones, which holds the potential translational benefit to these patients by targeting circulating histones and downstream pathways.