PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing.

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.

Neonatal injury models: integral tools to decipher the molecular basis of cardiac regeneration.

Myocardial injury often leads to heart failure due to the loss and insufficient regeneration of resident cardiomyocytes. The low regenerative potential of the mammalian heart is one of the main drivers of heart failure progression, especially after myocardial infarction accompanied by large contractile muscle loss. Preclinical therapies for cardiac regeneration are promising, but clinically still missing. Mammalian models represent an excellent translational in vivo platform to test drugs and treatments for the promotion of cardiac regeneration. Particularly, short-lived mice offer the possibility to monitor the outcome of such treatments throughout the life span. Importantly, there is a short period of time in newborn mice in which the heart retains full regenerative capacity after cardiac injury, which potentially also holds true for the neonatal human heart. Thus, in vivo neonatal mouse models of cardiac injury are crucial to gain insights into the molecular mechanisms underlying the cardiac regenerative processes and to devise novel therapeutic strategies for the treatment of diseased adult hearts. Here, we provide an overview of the established injury models to study cardiac regeneration. We summarize pioneering studies that demonstrate the potential of using neonatal cardiac injury models to identify factors that may stimulate heart regeneration by inducing endogenous cardiomyocyte proliferation in the adult heart. To conclude, we briefly summarize studies in large animal models and the insights gained in humans, which may pave the way toward the development of novel approaches in regenerative medicine.

Adult T-cells impair neonatal cardiac regeneration.

AIMS: Newborn mice and humans display transient cardiac regenerative potential that rapidly declines postnatally. Patients who survive a myocardial infarction (MI) often develop chronic heart failure due to the heart's poor regeneration capacity. We hypothesized that the cardiac 'regenerative-to-scarring' transition might be driven by the perinatal shifts observed in the circulating T-cell compartment. METHODS AND RESULTS: Post-MI immune responses were characterized in 1- (P1) vs. 7-day-old (P7) mice subjected to left anterior descending artery ligation. Myocardial infarction induced robust early inflammatory responses (36 h post-MI) in both age groups, but neonatal hearts exhibited rapid resolution of inflammation and full functional recovery. The perinatal loss of myocardial regenerative capacity was paralleled by a baseline increase in αß-T cell (CD4+ and CD8+) numbers. Strikingly, P1-infarcted mice reconstituted with adult T-cells shifted to an adult-like healing phenotype, marked by irreversible cardiac functional impairment and increased fibrosis. Infarcted neonatal mice harbouring adult T-cells also had more monocyte-derived macrophage recruitment, as typically seen in adults. At the transcriptome level, infarcted P1 hearts that received isolated adult T-cells showed enriched gene sets linked to fibrosis, inflammation, and interferon-gamma (IFN-γ) signalling. In contrast, newborn mice that received isolated Ifng-/- adult T-cells prior to MI displayed a regenerative phenotype that resembled that of its age-matched untreated controls. CONCLUSION: Physiological T-cell development or adoptive transfer of adult IFN-γ-producing T-cells into neonates contributed to impaired cardiac regeneration and promoted irreversible structural and functional cardiac damage. These findings reveal a trade-off between myocardial regenerative potential and the development of T-cell competence.

Cardiac regeneration: Options for repairing the injured heart.

Cardiac regeneration is one of the grand challenges in repairing injured human hearts. Numerous studies of signaling pathways and metabolism on cardiac development and disease pave the way for endogenous cardiomyocyte regeneration. New drug delivery approaches, high-throughput screening, as well as novel therapeutic compounds combined with gene editing will facilitate the development of potential cell-free therapeutics. In parallel, progress has been made in the field of cell-based therapies. Transplantation of human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs) can partially rescue the myocardial defects caused by cardiomyocyte loss in large animals. In this review, we summarize current cell-based and cell-free regenerative therapies, discuss the importance of cardiomyocyte maturation in cardiac regenerative medicine, and envision new ways of regeneration for the injured heart.

Predisposing and Precipitating Risk Factors for Delirium in Elderly Patients Admitted to a Cardiology Ward: An Observational Cohort Study in 1,042 Patients.

Aim: Although the risk factors for delirium in general medicine are well-established, their significance in cardiac diseases remains to be determined. Therefore, we evaluated the predisposing and precipitating risk factors in patients hospitalized with acute and chronic heart disease. Methods and Results: In this observational cohort study, 1,042 elderly patients (≥65 years) admitted to cardiology wards, 167 with and 875 without delirium, were included. The relevant sociodemographic and cardiac- and medical-related clusters were assessed by simple and multiple regression analyses and prediction models evaluating their association with delirium. The prevalence of delirium was 16.0%. The delirious patients were older (mean 80 vs. 76 years; p < 0.001) and more often institutionalized prior to admission (3.6 vs. 1.4%, p = 0.05), hospitalized twice as long (12 ± 10 days vs. 7 ± 7 days; p < 0.001), and discharged more often to nursing homes (4.8 vs. 0.6%, p < 0.001) or deceased (OR, 2.99; 95% CI, 1.53-5.85; p = 0.003). The most relevant risk factor was dementia (OR, 18.11; 95% CI, 5.77-56.83; p < 0.001), followed by history of stroke (OR, 6.61; 95% CI 1.35-32.44; p = 0.020), and pressure ulcers (OR, 3.62; 95% CI, 1.06-12.35; p = 0.040). The predicted probability for developing delirium was highest in patients with reduced mobility and institutionalization prior to admission (PP = 31.2%, p = 0.001). Of the cardiac diseases, only valvular heart disease (OR, 1.57; 95% CI, 1.01-2.44; p = 0.044) significantly predicted delirium. The patients undergoing cardiac interventions did not have higher rates of delirium (OR, 1.39; 95% CI 0.91-2.12; p = 0.124). Conclusion: In patients admitted to a cardiology ward, age-related functional and cognitive impairment, history of stroke, and pressure ulcers were the most relevant risk factors for delirium. With regards to specific cardiological factors, only valvular heart disease was associated with risk for delirium. Knowing these factors can help cardiologists to facilitate the early detection and management of delirium.

Endothelial dysfunction in COVID-19: Current findings and therapeutic implications.

Coronavirus disease 2019 (COVID-19) increases the risk of several non-pulmonary complications such as acute myocardial injury, renal failure or thromboembolic events. A possible unifying explanation for these phenomena may be the presence of profound endothelial dysfunction and injury. This review provides an overview on the association of endothelial dysfunction with COVID-19 and its therapeutic implications. Endothelial dysfunction is a common feature of the key comorbidities that increase risk for severe COVID-19 such as hypertension, obesity, diabetes mellitus, coronary artery disease or heart failure. Preliminary studies indicate that vascular endothelial cells can be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and evidence of widespread endothelial injury and inflammation is found in advanced cases of COVID-19. Prior evidence has established the crucial role of endothelial cells in maintaining and regulating vascular homeostasis and blood coagulation. Aggravation of endothelial dysfunction in COVID-19 may therefore impair organ perfusion and cause a procoagulatory state resulting in both macro- and microvascular thrombotic events. Angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs) and statins are known to improve endothelial dysfunction. Data from smaller observational studies and other viral infections suggests a possible beneficial effect in COVID-19. Other treatments that are currently under investigation for COVID-19 may also act by improving endothelial dysfunction in patients. Focusing therapies on preventing and improving endothelial dysfunction could improve outcomes in COVID-19. Several clinical trials are currently underway to explore this concept.

Cardioprotective effects of short-term empagliflozin treatment in db/db mice.

Sodium glucose transporter (SGLT)-2 inhibitors have consistently shown cardioprotective effects independent of the glycemic status of treated patients. In this study we aimed to investigate underlying mechanisms of short-term empagliflozin treatment in a mouse model of type II diabetes. Male db/db mice were fed a western type diet with or without enrichment with empagliflozin for 7 days. While glucose tolerance was significantly improved in empagliflozin treated mice, body weight and fasting insulin levels were comparable in both groups. Cardiac insulin signaling activity indicated by reduced proteinkinase B (AKT) phosphorylation was significantly decreased in the empagliflozin treated group. Remarkably, mitochondrial mass estimated by citrate synthase activity was significantly elevated in empagliflozin treated mice. Accordingly, mitochondrial morphology was significantly altered upon treatment with empagliflozin as analysed by transmission electron microscopy. Additionally, short-term empagliflozin therapy was associated with a changed cardiac tissue cytokine expression in favor of an anti-inflammatory pattern. Our data suggest that early cardioprotection in empagliflozin treated mice is independent of a reduction in body weight or hyperinsulinemia. Ameliorated mitochondrial ultrastructure, attenuated cardiac insulin signaling and diminished cardiac inflammation might contribute to the cardioprotective effects of empagliflozin.

Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy.

Angiogenesis is a hallmark of cancer, promoting growth and metastasis. Anti-angiogenic treatment has limited efficacy due to therapy-induced blood vessel alterations, often followed by local hypoxia, tumor adaptation, progression, and metastasis. It is therefore paramount to overcome therapy-induced resistance. We show that Apelin inhibition potently remodels the tumor microenvironment, reducing angiogenesis, and effectively blunting tumor growth. Functionally, targeting Apelin improves vessel function and reduces polymorphonuclear myeloid-derived suppressor cell infiltration. Importantly, in mammary and lung cancer, Apelin prevents resistance to anti-angiogenic receptor tyrosine kinase (RTK) inhibitor therapy, reducing growth and angiogenesis in lung and breast cancer models without increased hypoxia in the tumor microenvironment. Apelin blockage also prevents RTK inhibitor-induced metastases, and high Apelin levels correlate with poor prognosis of anti-angiogenic therapy patients. These data identify a druggable anti-angiogenic drug target that reduces tumor blood vessel densities and normalizes the tumor vasculature to decrease metastases.

Heart Failure Stimulates Tumor Growth by Circulating Factors.

BACKGROUND: Heart failure (HF) survival has improved, and nowadays, many patients with HF die of noncardiac causes, including cancer. Our aim was to investigate whether a causal relationship exists between HF and the development of cancer. METHODS: HF was induced by inflicting large anterior myocardial infarction in APCmin mice, which are prone to developing precancerous intestinal tumors, and tumor growth was measured. In addition, to rule out hemodynamic impairment, a heterotopic heart transplantation model was used in which an infarcted or sham-operated heart was transplanted into a recipient mouse while the native heart was left in situ. After 6 weeks, tumor number, volume, and proliferation were quantified. Candidate secreted proteins were selected because they were previously associated both with (colon) tumor growth and with myocardial production in post-myocardial infarction proteomic studies. Myocardial gene expression levels of these selected candidates were analyzed, as well as their proliferative effects on HT-29 (colon cancer) cells. We validated these candidates by measuring them in plasma of healthy subjects and patients with HF. Finally, we associated the relation between cardiac specific and inflammatory biomarkers and new-onset cancer in a large, prospective general population cohort. RESULTS: The presence of failing hearts, both native and heterotopically transplanted, resulted in significantly increased intestinal tumor load of 2.4-fold in APCmin mice (all P<0.0001). The severity of left ventricular dysfunction and fibrotic scar strongly correlated with tumor growth ( P=0.002 and P=0.016, respectively). We identified several proteins (including serpinA3 and A1, fibronectin, ceruloplasmin, and paraoxonase 1) that were elevated in human patients with chronic HF (n=101) compared with healthy subjects (n=180; P<0.001). Functionally, serpinA3 resulted in marked proliferation effects in human colon cancer (HT-29) cells, associated with Akt-S6 phosphorylation. Finally, elevated cardiac and inflammation biomarkers in apparently healthy humans (n=8319) were predictive of new-onset cancer (n=1124) independently of risk factors for cancer (age, smoking status, and body mass index). CONCLUSIONS: We demonstrate that the presence of HF is associated with enhanced tumor growth and that this is independent of hemodynamic impairment and could be caused by cardiac excreted factors. A diagnosis of HF may therefore be considered a risk factor for incident cancer.

Functionally Conserved Noncoding Regulators of Cardiomyocyte Proliferation and Regeneration in Mouse and Human.

BACKGROUND: The adult mammalian heart has little regenerative capacity after myocardial infarction (MI), whereas neonatal mouse heart regenerates without scarring or dysfunction. However, the underlying pathways are poorly defined. We sought to derive insights into the pathways regulating neonatal development of the mouse heart and cardiac regeneration post-MI. METHODS AND RESULTS: Total RNA-seq of mouse heart through the first 10 days of postnatal life (referred to as P3, P5, P10) revealed a previously unobserved transition in microRNA (miRNA) expression between P3 and P5 associated specifically with altered expression of protein-coding genes on the focal adhesion pathway and cessation of cardiomyocyte cell division. We found profound changes in the coding and noncoding transcriptome after neonatal MI, with evidence of essentially complete healing by P10. Over two-thirds of each of the messenger RNAs, long noncoding RNAs, and miRNAs that were differentially expressed in the post-MI heart were differentially expressed during normal postnatal development, suggesting a common regulatory pathway for normal cardiac development and post-MI cardiac regeneration. We selected exemplars of miRNAs implicated in our data set as regulators of cardiomyocyte proliferation. Several of these showed evidence of a functional influence on mouse cardiomyocyte cell division. In addition, a subset of these miRNAs, miR-144-3p, miR-195a-5p, miR-451a, and miR-6240 showed evidence of functional conservation in human cardiomyocytes. CONCLUSIONS: The sets of messenger RNAs, miRNAs, and long noncoding RNAs that we report here merit further investigation as gatekeepers of cell division in the postnatal heart and as targets for extension of the period of cardiac regeneration beyond the neonatal period.

A reproducible protocol for neonatal ischemic injury and cardiac regeneration in neonatal mice.

Cardiac regeneration is one of the prime visions in cardiovascular research. The mouse neonatal apical resection and left anterior descending artery (LAD) ligation model introduced novel in vivo mammalian assays to study cardiac regeneration. However, recent reports and editorials discussed and critically questioned the value and technical reproducibility of the mouse neonatal myocardial infarction approach, making it paramount to develop and use a reproducible model system. We established a mouse neonatal myocardial infarction model by visually confirmed ligation of the LAD using microsurgery. TdT-mediated dUTP nick-end labeling (TUNEL) proved reproducible massive myocardial infarctions in a defined region of the apex and anterior wall of neonatal and 7-day-old mice. Whereas hearts ligated on postnatal day 7 displayed chronic injury, cardiac samples ligated immediately after birth always showed complete structural regeneration after long-term follow-up. Cardiac regeneration was observed in all mouse stains (C57BL/6J, ICR, and mixed background C57BL/6JxSv129) tested so far. We present a detailed in vivo protocol to study complex mechanisms of complete cardiac repair following ischemic cardiac damage. Neonatal LAD ligation surgery is feasible, and results in reproducible myocardial infarctions 24 h after ligation, and no structural myocardial defects are detectable following long-term follow-up. We encourage the cardiovascular community to use our protocol and teaching video to answer key scientific questions in the field of cardiac regeneration.

A cryoinjury model in neonatal mice for cardiac translational and regeneration research.

The introduction of injury models for neonatal mouse hearts has accelerated research on the mechanisms of cardiac regeneration in mammals. However, some existing models, such as apical resection and ligation of the left anterior descending artery, produce variable results, which may be due to technical difficulties associated with these methods. Here we present an alternative model for the study of cardiac regeneration in neonatal mice in which cryoinjury is used to induce heart injury. This model yields a reproducible injury size, does not induce known mechanisms of cardiac regeneration and leads to a sustained reduction of cardiac function. This protocol uses reusable cryoprobes that can be assembled in 5 min, with the entire procedure taking 15 min per pup. The subsequent heart collection and fixation takes 2 d to complete. Cryoinjury results in a myocardial scar, and the size of injury can be scaled by the use of different cryoprobes (0.5 and 1.5 mm). Cryoinjury models are medically relevant to diseases in human infants with heart disease. In summary, the myocardial cryoinjury model in neonatal mice described here is a useful tool for cardiac translational and regeneration research.

Functional Recovery of a Human Neonatal Heart After Severe Myocardial Infarction.

RATIONALE: Cardiac remodeling and subsequent heart failure remain critical issues after myocardial infarction despite improved treatment and reperfusion strategies. Recently, cardiac regeneration has been demonstrated in fish and newborn mice after apex resection or cardiac infarctions. Two key issues remain to translate findings in model organisms to future therapies in humans: what is the mechanism and can cardiac regeneration indeed occur in newborn humans? OBJECTIVE: To assess whether human neonatal hearts can functionally recover after myocardial infarction. METHODS AND RESULTS: Here, we report the case of a newborn child having a severe myocardial infarction due to coronary artery occlusion. The child developed massive cardiac damage as defined by serum markers for cardiomyocyte cell death, electrocardiograms, echocardiography, and cardiac angiography. Remarkably, within weeks after the initial ischemic insult, we observed functional cardiac recovery, which translated into long-term normal heart function. CONCLUSIONS: These data indicate that, similar to neonatal rodents, newborn humans might have the intrinsic capacity to repair myocardial damage and completely recover cardiac function.

Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window.

Therapies developed for adult patients with heart failure have been shown to be ineffective in pediatric clinical trials, leading to the recognition that new pediatric-specific therapies for heart failure must be developed. Administration of the recombinant growth factor neuregulin-1 (rNRG1) stimulates regeneration of heart muscle cells (cardiomyocytes) in adult mice. Because proliferation-competent cardiomyocytes are more abundant in growing mammals, we hypothesized that administration of rNRG1 during the neonatal period might be more effective than in adulthood. If so, neonatal rNRG1 delivery could be a new therapeutic strategy for treating heart failure in pediatric patients. To evaluate the effectiveness of rNRG1 administration in cardiac regeneration, newborn mice were subjected to cryoinjury, which induced myocardial dysfunction and scar formation and decreased cardiomyocyte cell cycle activity. Early administration of rNRG1 to mice from birth to 34 days of age improved myocardial function and reduced the prevalence of transmural scars. In contrast, administration of rNRG1 from 4 to 34 days of age only transiently improved myocardial function. The mechanisms of early administration involved cardiomyocyte protection (38%) and proliferation (62%). We also assessed the ability of rNRG1 to stimulate cardiomyocyte proliferation in intact cultured myocardium from pediatric patients. rNRG1 induced cardiomyocyte proliferation in myocardium from infants with heart disease who were less than 6 months of age. Our results identify an effective time period within which to execute rNRG1 clinical trials in pediatric patients for the stimulation of cardiomyocyte regeneration.

In vivo cardiac role of migfilin during experimental pressure overload.

AIMS: Increased myocardial wall strain triggers the cardiac hypertrophic response by increasing cardiomyocyte size, reprogramming gene expression, and enhancing contractile protein synthesis. The LIM protein, migfilin, is a cytoskeleton-associated protein that was found to translocate in vitro into the nucleus in a Ca(2+)-dependent manner, where it co-activates the pivotal cardiac transcription factor Csx/Nkx2.5. However, the in vivo role of migfilin in cardiac function and stress response is unclear. METHODS AND RESULTS: To define the role of migfilin in cardiac hypertrophy, we induced hypertension by transverse aortic constriction (TAC) and compared cardiac morphology and function of migfilin knockout (KO) with wild-type (WT) hearts. Heart size and myocardial contractility were comparable in untreated migfilin KO and WT hearts, but migfilin-null hearts presented a reduced extent of hypertrophic remodelling in response to chronic hypertensile stress. Migfilin KO mice maintained their cardiac function for a longer time period compared with WT mice, which presented extensive fibrosis and death due to heart failure. Migfilin translocated into the nucleus of TAC-treated cardiomyocytes, and migfilin KO hearts showed reduced Akt activation during the early response to pressure overload. CONCLUSIONS: Our findings indicate an important role of migfilin in the regulation of cardiac hypertrophy upon experimental TAC.

Construction of a global pain systems network highlights phospholipid signaling as a regulator of heat nociception.

The ability to perceive noxious stimuli is critical for an animal's survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.

Complete cardiac regeneration in a mouse model of myocardial infarction.

Cardiac remodeling and subsequent heart failure remain critical issues after myocardial infarction despite improved treatment and reperfusion strategies. Recently, complete cardiac regeneration has been demonstrated in fish and newborn mice following resection of the cardiac apex. However, it remained entirely unclear whether the mammalian heart can also completely regenerate following a complex cardiac ischemic injury. We established a protocol to induce a severe heart attack in one-day-old mice using left anterior descending artery (LAD) ligation. LAD ligation triggered substantial cardiac injury in the left ventricle defined by Caspase 3 activation and massive cell death. Ischemia-induced cardiomyocyte death was also visible on day 4 after LAD ligation. Remarkably, 7 days after the initial ischemic insult, we observed complete cardiac regeneration without any signs of tissue damage or scarring. This tissue regeneration translated into long-term normal heart functions as assessed by echocardiography. In contrast, LAD ligations in 7-day-old mice resulted in extensive scarring comparable to adult mice, indicating that the regenerative capacity for complete cardiac healing after heart attacks can be traced to the first week after birth. RNAseq analyses of hearts on day 1, day 3, and day 10 and comparing LAD-ligated and sham-operated mice surprisingly revealed a transcriptional programme of major changes in genes mediating mitosis and cell division between days 1, 3 and 10 postnatally and a very limited set of genes, including genes regulating cell cycle and extracellular matrix synthesis, being differentially regulated in the regenerating hearts. We present for the first time a mammalian model of complete cardiac regeneration following a severe ischemic cardiac injury. This novel model system provides the unique opportunity to uncover molecular and cellular pathways that can induce cardiac regeneration after ischemic injury, findings that one day could be translated to human heart attack patients.