A Murine Model of Hyperlipidemia-Induced Heart Failure with Preserved Ejection Fraction.

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

Mechanistic basis for rescuing hypertrophic cardiomyopathy with myosin regulatory light chain phosphorylation.

We investigated the impact of the phosphomimetic (Ser15 → Asp15) myosin regulatory light chain (S15D-RLC) on the Super-Relaxed (SRX) state of myosin using previously characterized transgenic (Tg) S15D-D166V rescue mice, comparing them to the Hypertrophic Cardiomyopathy (HCM) Tg-D166V model and wild-type (WT) RLC mice. In the Tg-D166V model, we observed a disruption of the SRX state, resulting in a transition from SRX to DRX (Disordered Relaxed) state, which explains the hypercontractility of D166V-mutated myosin motors. The presence of the S15D moiety in Tg-S15D-D166V mice restored the SRX/DRX balance to levels comparable to Tg-WT, thus mitigating the hypercontractile behavior associated with the HCM-D166V mutation. Additionally, we investigated the impact of delivering the S15D-RLC molecule to the hearts of Tg-D166V mice via adeno-associated virus (AAV9) and compared their condition to AAV9-empty vector-injected or non-injected Tg-D166V animals. Tg-D166V mice injected with AAV9 S15D-RLC exhibited a significantly higher proportion of myosin heads in the SRX state compared to those injected with AAV9 empty vector or left non-injected. No significant effect was observed in Tg-WT hearts treated similarly. These findings suggest that AAV9-delivered phosphomimetic S15D-RLC modality mitigates the abnormal Tg-D166V phenotype without impacting the normal function of Tg-WT hearts. Global longitudinal strain analysis supported these observations, indicating that the S15D moiety can alleviate the HCM-D166V phenotype by restoring SRX stability and the SRX ↔ DRX equilibrium.

Efficacy of a growth hormone-releasing hormone agonist in a murine model of cardiometabolic heart failure with preserved ejection fraction.

Heart failure (HF) with preserved ejection fraction (HFpEF) represents a major unmet medical need owing to its diverse pathophysiology and lack of effective therapies. Potent synthetic, agonists (MR-356 and MR-409) of growth hormone-releasing hormone (GHRH) improve the phenotype of models of HF with reduced ejection fraction (HFrEF) and in cardiorenal models of HFpEF. Endogenous GHRH exhibits a broad range of regulatory influences in the cardiovascular (CV) system and aging and plays a role in several cardiometabolic conditions including obesity and diabetes. Whether agonists of GHRH can improve the phenotype of cardiometabolic HFpEF remains untested and unknown. Here we tested the hypothesis that MR-356 can mitigate/reverse the cardiometabolic HFpEF phenotype. C57BL6N mice received a high-fat diet (HFD) plus the nitric oxide synthase inhibitor (l-NAME) for 9 wk. After 5 wk of HFD + l-NAME regimen, animals were randomized to receive daily injections of MR-356 or placebo during a 4-wk period. Control animals received no HFD + l-NAME or agonist treatment. Our results showed the unique potential of MR-356 to treat several HFpEF-like features including cardiac hypertrophy, fibrosis, capillary rarefaction, and pulmonary congestion. MR-356 improved cardiac performance by improving diastolic function, global longitudinal strain (GLS), and exercise capacity. Importantly, the increased expression of cardiac pro-brain natriuretic peptide (pro-BNP), inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor-A (VEGF-A) was restored to normal levels suggesting that MR-356 reduced myocardial stress associated with metabolic inflammation in HFpEF. Thus, agonists of GHRH may be an effective therapeutic strategy for the treatment of cardiometabolic HFpEF phenotype.NEW & NOTEWORTHY This randomized study used rigorous hemodynamic tools to test the efficacy of a synthetic GHRH agonist to improve cardiac performance in a cardiometabolic HFpEF. Daily injection of the GHRH agonist, MR-356, reduced the HFpEF-like effects as evidenced by improved diastolic dysfunction, reduced cardiac hypertrophy, fibrosis, and pulmonary congestion. Notably, end-diastolic pressure and end-diastolic pressure-volume relationship were reset to control levels. Moreover, treatment with MR-356 increased exercise capacity and reduced myocardial stress associated with metabolic inflammation in HFpEF.

Synthetic growth hormone-releasing hormone agonist ameliorates the myocardial pathophysiology characteristic of heart failure with preserved ejection fraction.

AIMS: To test the hypothesis that the activation of the growth hormone-releasing hormone (GHRH) receptor signalling pathway within the myocardium both prevents and reverses diastolic dysfunction and pathophysiologic features consistent with heart failure with preserved ejection fraction (HFpEF). Impaired myocardial relaxation, fibrosis, and ventricular stiffness, among other multi-organ morbidities, characterize the phenotype underlying the HFpEF syndrome. Despite the rapidly increasing prevalence of HFpEF, few effective therapies have emerged. Synthetic agonists of the GHRH receptors reduce myocardial fibrosis, cardiomyocyte hypertrophy, and improve performance in animal models of ischaemic cardiomyopathy, independently of the growth hormone axis. METHODS AND RESULTS: CD1 mice received 4- or 8-week continuous infusion of angiotensin-II (Ang-II) to generate a phenotype with several features consistent with HFpEF. Mice were administered either vehicle or a potent synthetic agonist of GHRH, MR-356 for 4-weeks beginning concurrently or 4-weeks following the initiation of Ang-II infusion. Ang-II-treated animals exhibited diastolic dysfunction, ventricular hypertrophy, interstitial fibrosis, and normal ejection fraction. Cardiomyocytes isolated from these animals exhibited incomplete relaxation, depressed contractile responses, altered myofibrillar protein phosphorylation, and disturbed calcium handling mechanisms (ex vivo). MR-356 both prevented and reversed the development of the pathological phenotype in vivo and ex vivo. Activation of the GHRH receptors increased cAMP and cGMP in cardiomyocytes isolated from control animals but only cAMP in cardiac fibroblasts, suggesting that GHRH-A exert differential effects on cardiomyocytes and fibroblasts. CONCLUSION: These findings indicate that the GHRH receptor signalling pathway(s) represents a new molecular target to counteract dysfunctional cardiomyocyte relaxation by targeting myofilament phosphorylation and fibrosis. Accordingly, activation of GHRH receptors with potent, synthetic GHRH agonists may provide a novel therapeutic approach to management of the myocardial alterations associated with the HFpEF syndrome.

Hydroxychloroquine Mitigates Dilated Cardiomyopathy Phenotype in Transgenic D94A Mice.

In this study, we aimed to investigate whether short-term and low-dose treatment with hydroxychloroquine (HCQ), an antimalarial drug, can modulate heart function in a preclinical model of dilated cardiomyopathy (DCM) expressing the D94A mutation in cardiac myosin regulatory light chain (RLC) compared with healthy non-transgenic (NTg) littermates. Increased interest in HCQ came with the COVID-19 pandemic, but the risk of cardiotoxic side effects of HCQ raised concerns, especially in patients with an underlying heart condition, e.g., cardiomyopathy. Effects of HCQ treatment vs. placebo (H2O), administered in Tg-D94A vs. NTg mice over one month, were studied by echocardiography and muscle contractile mechanics. Global longitudinal strain analysis showed the HCQ-mediated improvement in heart performance in DCM mice. At the molecular level, HCQ promoted the switch from myosin's super-relaxed (SRX) to disordered relaxed (DRX) state in DCM-D94A hearts. This result indicated more myosin cross-bridges exiting a hypocontractile SRX-OFF state and assuming the DRX-ON state, thus potentially enhancing myosin motor function in DCM mice. This bottom-up investigation of the pharmacological use of HCQ at the level of myosin molecules, muscle fibers, and whole hearts provides novel insights into mechanisms by which HCQ therapy mitigates some abnormal phenotypes in DCM-D94A mice and causes no harm in healthy NTg hearts.

Mouse Model of Heart Failure With Preserved Ejection Fraction Driven by Hyperlipidemia and Enhanced Cardiac Low-Density Lipoprotein Receptor Expression.

Background The pathways of diastolic dysfunction and heart failure with preserved ejection fraction driven by lipotoxicity with metabolic syndrome are incompletely understood. Thus, there is an urgent need for animal models that accurately mimic the metabolic and cardiovascular phenotypes of this phenogroup for mechanistic studies. Methods and Results Hyperlipidemia was induced in WT-129 mice by 4 weeks of biweekly poloxamer-407 intraperitoneal injections with or without a single intravenous injection of adeno-associatedvirus 9-cardiac troponin T-low-density lipoprotein receptor (n=31), or single intravenous injection with adeno-associatedvirus 9-cardiac troponin T-low-density lipoprotein receptor alone (n=10). Treatment groups were compared with untreated or placebo controls (n=37). Echocardiography, blood pressure, whole-body plethysmography, ECG telemetry, activity wheel monitoring, and biochemical and histological changes were assessed at 4 to 8 weeks. At 4 weeks, double treatment conferred diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Blood pressure and whole-body plethysmography results were normal, but respiration decreased at 8 weeks (P<0.01). ECG and activity wheel monitoring, respectively, indicated heart block and decreased exercise activity (P<0.001). Double treatment promoted elevated myocardial lipids including total cholesterol, fibrosis, increased wet/dry lung (P<0.001) and heart weight/body weight (P<0.05). Xanthelasma, ascites, and cardiac ischemia were evident in double and single (p407) groups. Sudden death occurred between 6 and 12 weeks in double and single (p407) treatment groups. Conclusions We present a novel model of heart failure with preserved ejection fraction driven by dyslipidemia where mice acquire diastolic dysfunction, arrhythmia, cardiac hypertrophy, fibrosis, pulmonary congestion, exercise intolerance, and preserved ejection fraction in the absence of obesity, hypertension, kidney disease, or diabetes. The model can be applied to dissect pathways of metabolic syndrome that drive diastolic dysfunction in this lipotoxicity-mediated heart failure with preserved ejection fraction phenogroup mimic.

Col4a3-/- Mice on Balb/C Background Have Less Severe Cardiorespiratory Phenotype and SGLT2 Over-Expression Compared to 129x1/SvJ and C57Bl/6 Backgrounds.

Alport syndrome (AS) is a hereditary renal disorder with no etiological therapy. In the preclinical Col4a3-/- model of AS, disease progression and severity vary depending on mouse strain. The sodium-glucose cotransporter 2 (SGLT2) is emerging as an attractive therapeutic target in cardiac/renal pathologies, but its application to AS remains untested. This study investigates cardiorespiratory function and SGLT2 renal expression in Col4a3-/- mice from three different genetic backgrounds, 129x1/SvJ, C57Bl/6 and Balb/C. male Col4a3-/- 129x1/SvJ mice displayed alterations consistent with heart failure with preserved ejection fraction (HFpEF). Female, but not male, C57Bl/6 and Balb/C Col4a3-/- mice exhibited mild changes in systolic and diastolic function of the heart by echocardiography. Male C57Bl/6 Col4a3-/- mice presented systolic dysfunction by invasive hemodynamic analysis. All strains except Balb/C males demonstrated alterations in respiratory function. SGLT2 expression was significantly increased in AS compared to WT mice from all strains. However, cardiorespiratory abnormalities and SGLT2 over-expression were significantly less in AS Balb/C mice compared to the other two strains. Systolic blood pressure was significantly elevated only in mutant 129x1/SvJ mice. The results provide further evidence for strain-dependent cardiorespiratory and hypertensive phenotype variations in mouse AS models, corroborated by renal SGLT2 expression, and support ongoing initiatives to develop SGLT2 inhibitors for the treatment of AS.

S-Nitrosoglutathione Reductase Deficiency Causes Aberrant Placental S-Nitrosylation and Preeclampsia.

Background Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S-nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Methods and Results Here, we show that mice lacking S-nitrosoglutathione reductase (GSNOR-/-), a denitrosylase regulating protein S-nitrosylation, exhibit a preeclampsia phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. Reactive oxygen species, NO, and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental S-nitrosylated amino acid residues in GSNOR-/- mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S-nitrosoproteome, and identifies a unique set of S-nitrosylated proteins in GSNOR-/- mice. Importantly, human preeclamptic placentas exhibit decreased GSNOR activity and increased nitrosative stress. Conclusions Therefore, deficiency of GSNOR creates dysregulation of placental S-nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia.

Systemic delivery of large-scale manufactured Wharton's Jelly mesenchymal stem cell-derived extracellular vesicles improves cardiac function after myocardial infarction.

INTRODUCTION: Cardiovascular disease and myocardial infarction are leading causes of morbidity and mortality in aged populations. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are under evaluation as a therapeutic option for the treatment of myocardial infarction. AIM: This study aimed to develop a large-scale manufacturing procedure to harvest clinical-grade EVs required for the translation of EVs to the clinic. METHODS AND RESULTS: We compared the efficiency of large scale MSC-derived EV production and characterized EV miRNA cargo using the Quantum bioreactor with either fetal bovine serum or human platelet lysate (PLT)-containing expansion media. We tested the potency of the EV products in a murine model of acute myocardial infarction. Our results demonstrate an advantage of the Quantum bioreactor as a large-scale platform for EV production using PLT media; however, both media produced EVs with similar effects in vivo. The systemic delivery of EV products improved cardiac function following myocardial infarctions as indicated by a significant improvement in ejection fraction as well as parameters of cardiac performance, afterload, contractility and lusitropy. CONCLUSION: These findings have important implications for scale-up strategies of EVs and will facilitate clinical trials for their clinical evaluation.

Pigmentation Affects Elastic Fiber Patterning and Biomechanical Behavior of the Murine Aortic Valve.

The aortic valve (AoV) maintains unidirectional blood distribution from the left ventricle of the heart to the aorta for systemic circulation. The AoV leaflets rely on a precise extracellular matrix microarchitecture of collagen, elastin, and proteoglycans for appropriate biomechanical performance. We have previously demonstrated a relationship between the presence of pigment in the mouse AoV with elastic fiber patterning using multiphoton imaging. Here, we extended those findings using wholemount confocal microscopy revealing that elastic fibers were diminished in the AoV of hypopigmented mice (KitWv and albino) and were disorganized in the AoV of K5-Edn3 transgenic hyperpigmented mice when compared to wild type C57BL/6J mice. We further used atomic force microscopy to measure stiffness differences in the wholemount AoV leaflets of mice with different levels of pigmentation. We show that AoV leaflets of K5-Edn3 had overall higher stiffness (4.42 ± 0.35 kPa) when compared to those from KitWv (2.22 ± 0.21 kPa), albino (2.45 ± 0.16 kPa), and C57BL/6J (3.0 ± 0.16 kPa) mice. Despite the striking elastic fiber phenotype and noted stiffness differences, adult mutant mice were found to have no overt cardiac differences as measured by echocardiography. Our results indicate that pigmentation, but not melanocytes, is required for proper elastic fiber organization in the mouse AoV and dictates its biomechanical properties.

Growth hormone-releasing hormone agonists ameliorate chronic kidney disease-induced heart failure with preserved ejection fraction.

Therapies for heart failure with preserved ejection fraction (HFpEF) are lacking. Growth hormone-releasing hormone agonists (GHRH-As) have salutary effects in ischemic and nonischemic heart failure animal models. Accordingly, we hypothesized that GHRH-A treatment ameliorates chronic kidney disease (CKD)-induced HFpEF in a large-animal model. Female Yorkshire pigs (n = 16) underwent 5/6 nephrectomy via renal artery embolization and 12 wk later were randomized to receive daily subcutaneous injections of GHRH-A (MR-409; n = 8; 30 µg/kg) or placebo (n = 8) for 4 to 6 wk. Renal and cardiac structure and function were serially assessed postembolization. Animals with 5/6 nephrectomy exhibited CKD (elevated blood urea nitrogen [BUN] and creatinine) and faithfully recapitulated the hemodynamic features of HFpEF. HFpEF was demonstrated at 12 wk by maintenance of ejection fraction associated with increased left ventricular mass, relative wall thickness, end-diastolic pressure (EDP), end-diastolic pressure/end-diastolic volume (EDP/EDV) ratio, and tau, the time constant of isovolumic diastolic relaxation. After 4 to 6 wk of treatment, the GHRH-A group exhibited normalization of EDP (P = 0.03), reduced EDP/EDV ratio (P = 0.018), and a reduction in myocardial pro-brain natriuretic peptide protein abundance. GHRH-A increased cardiomyocyte [Ca2+] transient amplitude (P = 0.009). Improvement of the diastolic function was also evidenced by increased abundance of titin isoforms and their ratio (P = 0.0022). GHRH-A exerted a beneficial effect on diastolic function in a CKD large-animal model as demonstrated by improving hemodynamic, structural, and molecular characteristics of HFpEF. These findings have important therapeutic implications for the HFpEF syndrome.

Soluble Klotho, a biomarker and therapeutic strategy to reduce bronchopulmonary dysplasia and pulmonary hypertension in preterm infants.

Preterm infants with bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH) have accelerated lung aging and poor long-term outcomes. Klotho is an antiaging protein that modulates oxidative stress, angiogenesis and fibrosis. Here we test the hypothesis that decreased cord Klotho levels in preterm infants predict increased BPD-PH risk and early Klotho supplementation prevents BPD-like phenotype and PH in rodents exposed to neonatal hyperoxia. In experiment 1, Klotho levels were measured in cord blood of preterm infants who were enrolled in a longitudinal cohort study. In experiment 2, using an experimental BPD-PH model, rat pups exposed to room air or hyperoxia (85% O2) were randomly assigned to receive every other day injections of recombinant Klotho or placebo. The effect of Klotho on lung structure, PH and cardiac function was assessed. As compared to controls, preterm infants with BPD or BPD-PH had decreased cord Klotho levels. Early Klotho supplementation in neonatal hyperoxia-exposed rodents preserved lung alveolar and vascular structure, attenuated PH, reduced pulmonary vascular remodeling and improved cardiac function. Together, these findings have important implications as they suggest that perinatal Klotho deficiency contributes to BPD-PH risk and strategies that preserve Klotho levels, may improve long-term cardiopulmonary outcomes in preterm infants.

Ablation of the N terminus of cardiac essential light chain promotes the super-relaxed state of myosin and counteracts hypercontractility in hypertrophic cardiomyopathy mutant mice.

In this study, we focus on the molecular mechanisms associated with the A57G (Ala57-to-Gly57) mutation in myosin essential light chains (ELCs), found to cause hypertrophic cardiomyopathy (HCM) in humans and in mice. Specifically, we studied the effects of A57G on the super-relaxed (SRX) state of myosin that may contribute to the hypercontractile cross-bridge behavior and ultimately lead to pathological cardiac remodeling in transgenic Tg-A57G mice. The disease model was compared to Tg-WT mice, expressing the wild-type human ventricular ELC, and analyzed against Tg-Δ43 mice, expressing the N-terminally truncated ELC, whose hearts hypertrophy with time but do not show any abnormalities in cardiac morphology or function. Our data suggest a new role for the N terminus of cardiac ELC (N-ELC) in modulation of myosin cross-bridge function in the healthy as well as in HCM myocardium. The lack of N-ELC in Tg-Δ43 mice was found to significantly stabilize the SRX state of myosin and increase the number of myosin heads occupying a low-energy state. In agreement, Δ43 hearts showed significantly decreased ATP utilization and low actin-activated myosin ATPase compared with A57G and WT hearts. The hypercontractile activity of A57G-ELC cross-bridges was manifested by the inhibition of the SRX state, increased number of myosin heads available for interaction with actin, and higher ATPase activity. Fiber mechanics studies, echocardiography examination, and assessment of fibrosis confirmed the development of two distinct forms of cardiac remodeling in these two ELC mouse models, with pathological cardiac hypertrophy in Tg-A57G, and near physiologic cardiac growth in Tg-Δ43 animals.

Progenitor/Stem Cell Delivery by Suprarenal Aorta Route in Acute Kidney Injury.

Progenitor/stem cell-based kidney regenerative strategies are a key step towards the development of novel therapeutic regimens for kidney disease treatment. However, the route of cell delivery, e.g., intravenous, intra-arterial, or intra-parenchymal, may affect the efficiency for kidney repair in different models of acute and chronic injury. Here, we describe a protocol of intra-aorta progenitor/stem cell injection in rats following either acute ischemia-reperfusion injury or acute proteinuria induced by puromycin aminonucleoside (PAN) - the experimental prototype of human minimal change disease and early stages of focal and segmental glomerulosclerosis. Vascular clips were applied across both renal pedicles for 35 min, or a single dose of PAN was injected via intra-peritoneal route, respectively. Subsequently, 2 x 106 stem cells [green fluorescent protein (GFP)-labeled c-Kit+ progenitor/stem cells or GFP-mesenchymal stem cells] or saline were injected into the suprarenal aorta, above the renal arteries, after application of a vascular clip to the abdominal aorta below the renal arteries. This approach contributed to engraftment rates of ∼10% at day 8 post ischemia-reperfusion injury, when c-Kit+ progenitor/stem cells were injected, which accelerated kidney recovery. Similar rates of engraftment were found after PAN-induced podocyte damage at day 21. With practice and gentle surgical technique, 100% of the rats could be injected successfully, and, in the week following injection, ∼ 85% of the injected rats will recover completely. Given the similarities in mammals, much of the data obtained from intra-arterial delivery of progenitor/stem cells in rodents can be tested in translational research and clinical trials with endovascular catheters in humans.

Stabilization of the cardiac sarcolemma by sarcospan rescues DMD-associated cardiomyopathy.

In the current preclinical study, we demonstrate the therapeutic potential of sarcospan (SSPN) overexpression to alleviate cardiomyopathy associated with Duchenne muscular dystrophy (DMD) utilizing dystrophin-deficient mdx mice with utrophin haploinsufficiency that more accurately represent the severe disease course of human DMD. SSPN interacts with dystrophin, the DMD disease gene product, and its autosomal paralog utrophin, which is upregulated in DMD as a partial compensatory mechanism. SSPN transgenic mice have enhanced abundance of fully glycosylated α-dystroglycan, which may further protect dystrophin-deficient cardiac membranes. Baseline echocardiography reveals SSPN improves systolic function and hypertrophic indices in mdx and mdx:utr-heterozygous mice. Assessment of SSPN transgenic mdx mice by hemodynamic pressure-volume methods highlights enhanced systolic performance compared to mdx controls. SSPN restores cardiac sarcolemma stability, the primary defect in DMD disease, reduces fibrotic response and improves contractile function. We demonstrate that SSPN ameliorates more advanced cardiac disease in the context of diminished sarcolemma expression of utrophin and ß1D integrin that mitigate disease severity and partially restores responsiveness to ß-adrenergic stimulation. Overall, our current and previous findings suggest SSPN overexpression in DMD mouse models positively impacts skeletal, pulmonary and cardiac performance by addressing the stability of proteins at the sarcolemma that protect the heart from injury, supporting SSPN and membrane stabilization as a therapeutic target for DMD.

Therapeutic potential of AAV9-S15D-RLC gene delivery in humanized MYL2 mouse model of HCM.

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder characterized by ventricular hypertrophy, myofibrillar disarray, and fibrosis, and is primarily caused by mutations in sarcomeric genes. With no definitive cure for HCM, there is an urgent need for the development of novel preventive and reparative therapies. This study is focused on aspartic acid-to-valine (D166V) mutation in the myosin regulatory light chain, RLC (MYL2 gene), associated with a malignant form of HCM. Since myosin RLC phosphorylation is critical for normal cardiac function, we aimed to exploit this post-translational modification via phosphomimetic-RLC gene therapy. We hypothesized that mimicking/modulating cardiac RLC phosphorylation in non-phosphorylatable D166V myocardium would improve heart function of HCM-D166V mice. Adeno-associated virus, serotype-9 (AAV9) was used to deliver phosphomimetic human RLC variant with serine-to-aspartic acid substitution at Ser15-RLC phosphorylation site (S15D-RLC) into the hearts of humanized HCM-D166V mice. Improvement of heart function was monitored by echocardiography, invasive hemodynamics (PV-loops) and muscle contractile mechanics. A significant increase in cardiac output and stroke work and a decrease in relaxation constant, Tau, shown to be prolonged in HCM mice, were observed in AAV- vs. PBS-injected HCM mice. Strain analysis showed enhanced myocardial longitudinal shortening in AAV-treated vs. control mice. In addition, increased maximal contractile force was observed in skinned papillary muscles from AAV-injected HCM hearts. Our data suggest that myosin RLC phosphorylation may have important translational implications for the treatment of RLC mutations-induced HCM and possibly play a role in other disease settings accompanied by depressed Ser15-RLC phosphorylation. KEY MESSAGES: HCM-D166V mice show decreased RLC phosphorylation and decompensated function. AAV9-S15D-RLC gene therapy in HCM-D166V mice, but not in WT-RLC, results in improved heart performance. Global longitudinal strain analysis shows enhanced contractility in AAV vs controls. Increased systolic and diastolic function is paralleled by higher contractile force. Phosphomimic S15D-RLC has a therapeutic potential for HCM.

Kidney-derived c-kit+ progenitor/stem cells contribute to podocyte recovery in a model of acute proteinuria.

Kidney-derived c-kit+ cells exhibit progenitor/stem cell properties and can regenerate epithelial tubular cells following ischemia-reperfusion injury in rats. We therefore investigated whether c-kit+ progenitor/stem cells contribute to podocyte repair in a rat model of acute proteinuria induced by puromycin aminonucleoside (PAN), the experimental prototype of human minimal change disease and early stages of focal and segmental glomerulosclerosis. We found that c-kit+ progenitor/stem cells accelerated kidney recovery by improving foot process effacement (foot process width was lower in c-kit group vs saline treated animals, P = 0.03). In particular, these cells engrafted in small quantity into tubules, vessels, and glomeruli, where they occasionally differentiated into podocyte-like cells. This effect was related to an up regulation of α-Actinin-4 and mTORC2-Rictor pathway. Activation of autophagy by c-kit+ progenitor/stem cells also contributed to kidney regeneration and intracellular homeostasis (autophagosomes and autophagolysosomes number and LC3A/B-I and LC3A/B-II expression were higher in the c-kit group vs saline treated animals, P = 0.0031 and P = 0.0009, respectively). Taken together, our findings suggest that kidney-derived c-kit+ progenitor/stem cells exert reparative effects on glomerular disease processes through paracrine effects, to a lesser extent differentiation into podocyte-like cells and contribution to maintenance of podocyte cytoskeleton after injury. These findings have clinical implications for cell therapy of glomerular pathobiology.

Hypercontractile mutant of ventricular myosin essential light chain leads to disruption of sarcomeric structure and function and results in restrictive cardiomyopathy in mice.

AIMS: The E143K (Glu → Lys) mutation in the myosin essential light chain has been associated with restrictive cardiomyopathy (RCM) in humans, but the mechanisms that underlie the development of defective cardiac function are unknown. Using transgenic E143K-RCM mice, we sought to determine the molecular and cellular triggers of E143K-induced heart remodelling. METHODS AND RESULTS: The E143K-induced abnormalities in cardiac function and morphology observed by echocardiography and invasive haemodynamics were paralleled by augmented active and passive tension measured in skinned papillary muscle fibres compared with wild-type (WT)-generated force. In vitro, E143K-myosin had increased duty ratio and binding affinity to actin compared with WT-myosin, increased actin-activated ATPase activity and slower rates of ATP-dependent dissociation of the acto-myosin complex, indicating an E143K-induced myosin hypercontractility. E143K was also observed to reduce the level of myosin regulatory light chain phosphorylation while that of troponin-I remained unchanged. Small-angle X-ray diffraction data showed a decrease in the filament lattice spacing (d1,0) with no changes in the equatorial reflections intensity ratios (I1,1/I1,0) in E143K vs. WT skinned papillary muscles. The hearts of mutant-mice demonstrated ultrastructural defects and fibrosis that progressively worsened in senescent animals and these changes were hypothesized to contribute to diastolic disturbance and to mild systolic dysfunction. Gene expression profiles of E143K-hearts supported the histopathology results and showed an upregulation of stress-response and collagen genes. Finally, proteomic analysis evidenced RCM-dependent metabolic adaptations and higher energy demands in E143K vs. WT hearts. CONCLUSIONS: As a result of the E143K-induced myosin hypercontractility, the hearts of RCM mice model exhibited cardiac dysfunction, stiff ventricles and physiological, morphologic, and metabolic remodelling consistent with the development of RCM. Future efforts should be directed toward normalization of myosin motor function and the use of myosin-specific therapeutics to avert the hypercontractile state of E143K-myosin and prevent pathological cardiac remodelling.

cKit+ cardiac progenitors of neural crest origin.

The degree to which cKit-expressing progenitors generate cardiomyocytes in the heart is controversial. Genetic fate-mapping studies suggest minimal contribution; however, whether or not minimal contribution reflects minimal cardiomyogenic capacity is unclear because the embryonic origin and role in cardiogenesis of these progenitors remain elusive. Using high-resolution genetic fate-mapping approaches with cKit(CreERT2/+) and Wnt1::Flpe mouse lines, we show that cKit delineates cardiac neural crest progenitors (CNC(kit)). CNC(kit) possess full cardiomyogenic capacity and contribute to all CNC derivatives, including cardiac conduction system cells. Furthermore, by modeling cardiogenesis in cKit(CreERT2)-induced pluripotent stem cells, we show that, paradoxically, the cardiogenic fate of CNC(kit) is regulated by bone morphogenetic protein antagonism, a signaling pathway activated transiently during establishment of the cardiac crescent, and extinguished from the heart before CNC invasion. Together, these findings elucidate the origin of cKit(+) cardiac progenitors and suggest that a nonpermissive cardiac milieu, rather than minimal cardiomyogenic capacity, controls the degree of CNC(kit) contribution to myocardium.

Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice.

Myosin light chain kinase (MLCK)-dependent phosphorylation of the regulatory light chain (RLC) of cardiac myosin is known to play a beneficial role in heart disease, but the idea of a phosphorylation-mediated reversal of a hypertrophic cardiomyopathy (HCM) phenotype is novel. Our previous studies on transgenic (Tg) HCM-RLC mice revealed that the D166V (Aspartate166 → Valine) mutation-induced changes in heart morphology and function coincided with largely reduced RLC phosphorylation in situ. We hypothesized that the introduction of a constitutively phosphorylated Serine15 (S15D) into the hearts of D166V mice would prevent the development of a deleterious HCM phenotype. In support of this notion, MLCK-induced phosphorylation of D166V-mutated hearts was found to rescue some of their abnormal contractile properties. Tg-S15D-D166V mice were generated with the human cardiac RLC-S15D-D166V construct substituted for mouse cardiac RLC and were subjected to functional, structural, and morphological assessments. The results were compared with Tg-WT and Tg-D166V mice expressing the human ventricular RLC-WT or its D166V mutant, respectively. Echocardiography and invasive hemodynamic studies demonstrated significant improvements of intact heart function in S15D-D166V mice compared with D166V, with the systolic and diastolic indices reaching those monitored in WT mice. A largely reduced maximal tension and abnormally high myofilament Ca(2+) sensitivity observed in D166V-mutated hearts were reversed in S15D-D166V mice. Low-angle X-ray diffraction study revealed that altered myofilament structures present in HCM-D166V mice were mitigated in S15D-D166V rescue mice. Our collective results suggest that expression of pseudophosphorylated RLC in the hearts of HCM mice is sufficient to prevent the development of the pathological HCM phenotype.