Characterization of heterozygous and homozygous mouse models with the most common hypertrophic cardiomyopathy mutation MYBPC3 c.2373InsG in the Netherlands.

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in the cardiac myosin binding protein-C (cMyBP-C) encoding gene MYBPC3. In the Netherlands, approximately 25% of patients carry the MYBPC3 c.2373InsG founder mutation. Most patients are heterozygous (MYBPC3 +/InsG) and have highly variable phenotypic expression, whereas homozygous (MYBPC3 InsG/InsG) patients have severe HCM at a young age. To improve understanding of disease progression and genotype-phenotype relationship based on the hallmarks of human HCM, we characterized mice with CRISPR/Cas9-induced heterozygous and homozygous mutations. At 18-28 weeks of age, we assessed the cardiac phenotype of Mybpc3 +/InsG and Mybpc3 InsG/InsG mice with echocardiography, and performed histological analyses. Cytoskeletal proteins and cardiomyocyte contractility of 3-4 week old and 18-28 week old Mybpc3 c.2373InsG mice were compared to wild-type (WT) mice. Expectedly, knock-in of Mybpc3 c.2373InsG resulted in the absence of cMyBP-C and our 18-28 week old homozygous Mybpc3 c.2373InsG model developed cardiac hypertrophy and severe left ventricular systolic and diastolic dysfunction, whereas HCM was not evident in Mybpc3 +/InsG mice. Mybpc3 InsG/InsG cardiomyocytes also presented with slowed contraction-relaxation kinetics, to a greater extent in 18-28 week old mice, partially due to increased levels of detyrosinated tubulin and desmin, and reduced cardiac troponin I (cTnI) phosphorylation. Impaired cardiomyocyte contraction-relaxation kinetics were successfully normalized in 18-28 week old Mybpc3 InsG/InsG cardiomyocytes by combining detyrosination inhibitor parthenolide and β-adrenergic receptor agonist isoproterenol. Both the 3-4 week old and 18-28 week old Mybpc3 InsG/InsG models recapitulate HCM, with a severe phenotype present in the 18-28 week old model

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy

BACKGROUND: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. METHODS: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. RESULTS: Screening of 157 kinase inhibitors in wild-type (N=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (N=24 HCM and N=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (N=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. CONCLUSIONS: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.</p

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4–8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions

Single dose of empagliflozin increases in vivo cardiac energy status in diabetic db/db mice

In the EMPA-REG OUTCOME trial, empagliflozin, a potent and specific inhibitor of the sodium glucose co-transporter 2, showed impressive benefits on cardiovascular outcome in patients with Type 2 diabetes.1 Empagliflozin reduced three-point primary composite outcome (cardiovascular death, non-fatal myocardial infarction, or non-fatal stroke) by 14%, which was mainly attributed to a 38% relative risk reduction in cardiovascular death.1 A 35% relative risk reduction in hospitalization for heart failure and a 32% relative risk reduction in all-cause mortality was also reported.1 However, the underlying mechanisms explaining these beneficial outcomes are yet to be elucidated. Deprivation of cardiac energy, characterized by a decreased cardiac phosphocreatine-to-ATP ratio (PCr/ATP), has been proposed to play a major role in the development of heart failure.2 Empagliflozin increases plasma ketone body levels and it has therefore been hypothesized that a shift in energy substrate metabolism towards ketones or an increased availability of ketones as add-on fuel could explain the positive cardiovascular outcomes in the EMPA-REG study.3 To test the ‘fuel hypothesis’, we investigated whether an increase in plasma ketones by empagliflozin was accompanied by an increase in cardiac PCr/ATP. We administered a single dose of empagliflozin in fasting db/db mice, to simulate a situation in which plasma ketone levels are immediately increased. This acute experimental design allows investigating the effect of alterations in fuel availability on changes in cardiac PCr/ATP ratio without interference from other factors, such as cardiac remodelling after long-term treatment. Using 31P magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI), we measured in vivo cardiac PCr/ATP and function, respectively

Single dose of empagliflozin increases in vivo cardiac energy status in diabetic db/db mice

In the EMPA-REG OUTCOME trial, empagliflozin, a potent and specific inhibitor of the sodium glucose co-transporter 2, showed impressive benefits on cardiovascular outcome in patients with Type 2 diabetes.1 Empagliflozin reduced three-point primary composite outcome (cardiovascular death, non-fatal myocardial infarction, or non-fatal stroke) by 14%, which was mainly attributed to a 38% relative risk reduction in cardiovascular death.1 A 35% relative risk reduction in hospitalization for heart failure and a 32% relative risk reduction in all-cause mortality was also reported.1 However, the underlying mechanisms explaining these beneficial outcomes are yet to be elucidated. Deprivation of cardiac energy, characterized by a decreased cardiac phosphocreatine-to-ATP ratio (PCr/ATP), has been proposed to play a major role in the development of heart failure.2 Empagliflozin increases plasma ketone body levels and it has therefore been hypothesized that a shift in energy substrate metabolism towards ketones or an increased availability of ketones as add-on fuel could explain the positive cardiovascular outcomes in the EMPA-REG study.3 To test the ‘fuel hypothesis’, we investigated whether an increase in plasma ketones by empagliflozin was accompanied by an increase in cardiac PCr/ATP. We administered a single dose of empagliflozin in fasting db/db mice, to simulate a situation in which plasma ketone levels are immediately increased. This acute experimental design allows investigating the effect of alterations in fuel availability on changes in cardiac PCr/ATP ratio without interference from other factors, such as cardiac remodelling after long-term treatment. Using 31P magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI), we measured in vivo cardiac PCr/ATP and function, respectively

Single dose of empagliflozin increases in vivo cardiac energy status in diabetic db/db mice

In the EMPA-REG OUTCOME trial, empagliflozin, a potent and specific inhibitor of the sodium glucose co-transporter 2, showed impressive benefits on cardiovascular outcome in patients with Type 2 diabetes.1 Empagliflozin reduced three-point primary composite outcome (cardiovascular death, non-fatal myocardial infarction, or non-fatal stroke) by 14%, which was mainly attributed to a 38% relative risk reduction in cardiovascular death.1 A 35% relative risk reduction in hospitalization for heart failure and a 32% relative risk reduction in all-cause mortality was also reported.1 However, the underlying mechanisms explaining these beneficial outcomes are yet to be elucidated. Deprivation of cardiac energy, characterized by a decreased cardiac phosphocreatine-to-ATP ratio (PCr/ATP), has been proposed to play a major role in the development of heart failure.2 Empagliflozin increases plasma ketone body levels and it has therefore been hypothesized that a shift in energy substrate metabolism towards ketones or an increased availability of ketones as add-on fuel could explain the positive cardiovascular outcomes in the EMPA-REG study.3\u3cbr/\u3e\u3cbr/\u3eTo test the ‘fuel hypothesis’, we investigated whether an increase in plasma ketones by empagliflozin was accompanied by an increase in cardiac PCr/ATP. We administered a single dose of empagliflozin in fasting db/db mice, to simulate a situation in which plasma ketone levels are immediately increased. This acute experimental design allows investigating the effect of alterations in fuel availability on changes in cardiac PCr/ATP ratio without interference from other factors, such as cardiac remodelling after long-term treatment. Using 31P magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI), we measured in vivo cardiac PCr/ATP and function, respectively

Muscle weakness in a S. pneumoniae sepsis mouse model

Background: The pathophysiology of intensive care unit-acquired weakness (ICU-AW), which affects peripheral nerves, limb muscles and respiratory muscles, is complex and incompletely understood. This illustrates the need for an ICU-AW animal model. However, a translatable and easily applicable ICU-AW animal model does not exist. The objective of this study was to investigate whether induction of a S. pneumoniae sepsis could serve as a model for ICU-AW. Methods: A total of 24 C57BL/6J mice were infected intranasally with viable S. pneumoniae. Control mice (n=8) received intranasal saline and mice of the blank group (n=4) were not inoculated. Ceftriaxone was administered at 24 h (n=8) or at 48h after inoculation (n=8), or as soon as mice lost 10% of their body weight (n=8). The primary endpoint, in vivo grip strength, was measured daily. At the end of the experiment, at 120 h after inoculation, electrophysiological recordings were performed and diaphragm muscle was excised to determine ex vivo muscle fiber strength and myosin/action ratio. Results: Grip strength over time was similar between experimental and control groups and electrophysiological recordings did not show signs of ICU-AW. Diaphragm fiber contractility measurements showed reduced strength in the group that received ceftriaxone at 48 h after S. pneumoniae inoculation. Conclusions: Ex vivo diaphragm weakness, but no in vivo limb weakness was found in the S. pneumoniae mouse model in which severe illness was induced. This does not reflect the full clinical picture of ICU-AW as seen in humans and as such this model did not fulfill our predefined requirements. However, this model may be used to study inflammation induced diaphragmatic weakness

The striated muscles in pulmonary arterial hypertension: adaptations beyond the right ventricle

Pulmonary arterial hypertension (PAH) is a fatal lung disease characterised by progressive remodelling of the small pulmonary vessels. The daily-life activities of patients with PAH are severely limited by exertional fatigue and dyspnoea. Typically, these symptoms have been explained by right heart failure. However, an increasing number of studies reveal that the impact of the PAH reaches further than the pulmonary circulation. Striated muscles other than the right ventricle are affected in PAH, such as the left ventricle, the diaphragm and peripheral skeletal muscles. Alterations in these striated muscles are associated with exercise intolerance and reduced quality of life. In this Back to Basics article on striated muscle function in PAH, we provide insight into the pathophysiological mechanisms causing muscle dysfunction in PAH and discuss potential new therapeutic strategies to restore muscle dysfunction

Large-Scale Contractility Measurements Reveal Large Atrioventricular and Subtle Interventricular Differences in Cultured Unloaded Rat Cardiomyocytes

The chambers of the heart fulfill different hemodynamic functions, which are reflected in their structural and contractile properties. While the atria are highly elastic to allow filling from the venous system, the ventricles need to be able to produce sufficiently high pressures to eject blood into the circulation. The right ventricle (RV) pumps into the low pressure pulmonary circulation, while the left ventricle (LV) needs to overcome the high pressure of the systemic circulation. It is incompletely understood whether these differences can be explained by the contractile differences at the level of the individual cardiomyocytes of the chambers. We addressed this by isolating cardiomyocytes from atria, RV, LV, and interventricular septum (IVS) of five healthy wild-type rats. Using a high-throughput contractility set-up, we measured contractile function of 2,043 cells after overnight culture. Compared to ventricular cardiomyocytes, atrial cells showed a twofold lower contraction amplitude and 1.4- to 1.7-fold slower kinetics of contraction and relaxation. The interventricular differences in contractile function were much smaller; RV cells displayed 12–13% less fractional shortening and 5–9% slower contraction and 3–15% slower relaxation kinetics relative to their LV and IVS counterparts. Aided by a large dataset, we established relationships between contractile parameters and found contraction velocity, fractional shortening and relaxation velocity to be highly correlated. In conclusion, our findings are in line with contractile differences observed at the atrioventricular level, but can only partly explain the interventricular differences that exist at the organ level