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.

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.

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.

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.

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.

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.

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.

S-Nitrosoglutathione Reductase Deficiency Enhances the Proliferative Expansion of Adult Heart Progenitors and Myocytes Post Myocardial Infarction.

BACKGROUND: Mammalian heart regenerative activity is lost before adulthood but increases after cardiac injury. Cardiac repair mechanisms, which involve both endogenous cardiac stem cells (CSCs) and cardiomyocyte cell-cycle reentry, are inadequate to achieve full recovery after myocardial infarction (MI). Mice deficient in S-nitrosoglutathione reductase (GSNOR(-/-)), an enzyme regulating S-nitrosothiol turnover, have preserved cardiac function after MI. Here, we tested the hypothesis that GSNOR activity modulates cardiac cell proliferation in the post-MI adult heart. METHODS AND RESULTS: GSNOR(-/-) and C57Bl6/J (wild-type [WT]) mice were subjected to sham operation (n=3 GSNOR(-/-); n=3 WT) or MI (n=41 GSNOR(-/-); n=65 WT). Compared with WT, GSNOR(-/-) mice exhibited improved survival, cardiac performance, and architecture after MI, as demonstrated by higher ejection fraction (P<0.05), lower endocardial volumes (P<0.001), and diminished scar size (P<0.05). In addition, cardiomyocytes from post-MI GSNOR(-/-) hearts exhibited faster calcium decay and sarcomeric relaxation times (P<0.001). Immunophenotypic analysis illustrated that post-MI GSNOR(-/-) hearts demonstrated enhanced neovascularization (P<0.001), c-kit(+) CSC abundance (P=0.013), and a ≈3-fold increase in proliferation of adult cardiomyocytes and c-kit(+)/CD45(-) CSCs (P<0.0001 and P=0.023, respectively) as measured by using 5-bromodeoxyuridine. CONCLUSIONS: Loss of GSNOR confers enhanced post-MI cardiac regenerative activity, characterized by enhanced turnover of cardiomyocytes and CSCs. Endogenous denitrosylases exert an inhibitory effect over cardiac repair mechanisms and therefore represents a potential novel therapeutic target.

New therapeutic approach to heart failure due to myocardial infarction based on targeting growth hormone-releasing hormone receptor.

BACKGROUND: We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective following myocardial infarction (MI). Here, our aim was to evaluate the in vitro and in vivo activities of highly potent new GHRH agonists, and elucidate their mechanisms of action in promoting cardiac repair. METHODS AND RESULTS: H9c2 cells were cultured in serum-free medium, mimicking nutritional deprivation. GHRH agonists decreased calcium influx and significantly improved cell survival. Rats with cardiac infarction were treated with GHRH agonists or placebo for four weeks. MI size was reduced by selected GHRH agonists (JI-38, MR-356, MR-409); this accompanied an increased number of cardiac c-kit+ cells, cellular mitotic divisions, and vascular density. One week post-MI, MR-409 significantly reduced plasma levels of IL-2, IL-6, IL-10 and TNF-α compared to placebo. Gene expression studies revealed favorable outcomes of MR-409 treatment partially result from inhibitory activity on pro-apoptotic molecules and pro-fibrotic systems, and by elevation of bone morphogenetic proteins. CONCLUSIONS: Treatment with GHRH agonists appears to reduce the inflammatory responses post-MI and may consequently improve mechanisms of healing and cardiac remodeling by regulating pathways involved in fibrosis, apoptosis and cardiac repair. Patients with cardiac dysfunction could benefit from treatment with novel GHRH agonists.

Agonists of growth hormone-releasing hormone stimulate self-renewal of cardiac stem cells and promote their survival.

The beneficial effects of agonists of growth hormone-releasing hormone receptor (GHRH-R) in heart failure models are associated with an increase in the number of ckit(+) cardiac stem cells (CSCs). The goal of the present study was to determine the presence of GHRH-R in CSCs, the effect of GHRH-R agonists on their proliferation and survival, and the mechanisms involved. We investigated the expression of GHRH-R in CSCs of different species and the effect of GHRH-R agonists on their cell proliferation and survival. GHRH-R is expressed in ckit(+) CSCs isolated from mouse, rat, and pig. Treatment of porcine CSCs with the GHRH-R agonist JI-38 significantly increased the rate of cell division. Similar results were observed with other GHRH-R agonists, MR-356 and MR-409. JI-38 exerted a protective effect on survival of porcine CSCs under conditions of oxidative stress induced by exposure to hydrogen peroxide. Treatment with JI-38 before exposure to peroxide significantly reduced cell death. A similar effect was observed with MR-356. Addition of GHRH-R agonists to porcine CSCs induced activation of ERK and AKT pathways as determined by increased expression of phospho-ERK and phospho-AKT. Inhibitors of ERK and AKT pathways completely reversed the effect of GHRH-R agonists on CSC proliferation. Our findings extend the observations of the expression of GHRH-R by CSCs and demonstrate that GHRH-R agonists have a direct effect on proliferation and survival of CSCs. These results support the therapeutic use of GHRH-R agonists for stimulating endogenous mechanisms for myocardial repair or for preconditioning of stem cells before transplantation.

Synthesis of new potent agonistic analogs of growth hormone-releasing hormone (GHRH) and evaluation of their endocrine and cardiac activities.

In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1-29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. "Agmatine analogs", MR-356 (N-Me-Tyr(1)-JI-38), MR-361(N-Me-Tyr(1), D-Ala(2)-JI-38) and MR-367(N-Me-Tyr(1), D-Ala(2), Asn(8)-JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr(1), showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr(1) and Arg(29)-NHCH3 as in MR-403 (N-Me-Tyr(1), D-Ala(2), Arg(29)-NHCH3-JI-38), MR-406 (N-Me-Tyr(1), Arg(29)-NHCH3-JI-38) and MR-409 (N-Me-Tyr(1), D-Ala(2), Asn(8), Arg(29)-NHCH3-JI-38), and MR-410 (N-Me-Tyr(1), D-Ala(2), Thr(8), Arg(29)-NHCH3-JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa(30)-NH2 such as MR-326 (N-Me-Tyr(1), D-Ala(2), Arg(29), Apa(30)-NH2-JI-38), and with Gab(30)-NH2, as MR-502 (D-Ala(2), 5F-Phe(6), Ser(28), Arg(29),Gab(30)-NH2-JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.

C-kit(+) cells isolated from developing kidneys are a novel population of stem cells with regenerative potential.

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit(+) cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit(+) cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex vivo expanded c-kit(+) cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together, these findings document a novel neonatal rat kidney c-kit(+) stem cell population that can be isolated, expanded, cloned, differentiated, and used for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications.

Pharmacologic and genetic strategies to enhance cell therapy for cardiac regeneration.

Cell-based therapy is emerging as an exciting potential therapeutic approach for cardiac regeneration following myocardial infarction (MI). As heart failure (HF) prevalence increases over time, development of new interventions designed to aid cardiac recovery from injury are crucial and should be considered more broadly. In this regard, substantial efforts to enhance the efficacy and safety of cell therapy are continuously growing along several fronts, including modifications to improve the reprogramming efficiency of inducible pluripotent stem cells (iPS), genetic engineering of adult stem cells, and administration of growth factors or small molecules to activate regenerative pathways in the injured heart. These interventions are emerging as potential therapeutic alternatives and/or adjuncts based on their potential to promote stem cell homing, proliferation, differentiation, and/or survival. Given the promise of therapeutic interventions to enhance the regenerative capacity of multipotent stem cells as well as specifically guide endogenous or exogenous stem cells into a cardiac lineage, their application in cardiac regenerative medicine should be the focus of future clinical research. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure."

Effects of combination of proliferative agents and erythropoietin on left ventricular remodeling post-myocardial infarction.

UNLABELLED: Erythropoietin (EPO) has the potential to improve ischemic tissue by mobilizing endothelial progenitor cells and enhancing neovascularization. We hypothesized that combining EPO with human chorionic gonadotrophin (hCG) would improve post-myocardial infarction (MI) effects synergistically. METHODS: After MI, five to seven animals were randomly assigned to each of the following treatments: control; hCG; EPO; hCG + EPO, and prolactin (PRL) + EPO. Follow-up echocardiograms were performed to assess cardiac structure and function. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and western blot analysis for apoptosis-related proteins, and cell proliferation by immunostaining for Ki67 and c-kit cells. RESULTS: The MI-mediated increased chamber systolic dimension (p < 0.05 in controls) was attenuated by hCG, EPO, and hCG + EPO (p < 0.05 vs. control) but not PRL + EPO. Similarly all treatment groups, except PRL + EPO, reduced MI-induced increases (p < 0.05 vs. control) in ejection fraction (EF). The functional improvement in the EPO-treated groups was accompanied by increased capillary density. Apoptosis was markedly reduced in all treated groups. Significantly more cardiac c-kit(+) cells were found in the hCG + EPO group. CONCLUSION: Our findings revealed that EPO, hCG, or their combination ameliorate cardiac remodeling post-MI. Whereas EPO stimulates neovascularization only and hCG + EPO stimulates c-kit+ cell proliferation. These data suggest that combining mobilizing and proliferative agents adds to the durability and sustainability of cytokine-based therapies for remodeling post-MI.

Sex-specific impact of aldosterone receptor antagonism on ventricular remodeling and gene expression after myocardial infarction.

Aldosterone receptor antagonism reduces mortality and improves post-myocardial infarction (MI) remodeling. Because aldosterone and estrogen signaling pathways interact, we hypothesized that aldosterone blockade is sex-specific. Therefore, we investigated the impact of eplerenone on left ventricular (LV) remodeling and gene expression of male infarcted rats versus female infarcted rats. MI and Sham animals were randomized to receive eplerenone (100 mg/kg/day) or placebo 3 days post-surgery for 4 weeks and assessed by echocardiography. In the MI placebo group, left ventricular end-diastolic dimension (LVEDD) increased from 7.3 +/- 0.4 mm to 10.2 +/- 1.0 mm (p < 0.05) and ejection fraction (EF) decreased from 82.3 +/- 4% to 45.5 +/- 11% (p < 0.05) in both sexes (p = NS between groups). Eplerenone attenuated LVEDD enlargement more effectively in females (8.8 +/- 0.2 mm, p < 0.05 vs. placebo) than in males (9.7 +/- 0.2 mm, p = NS vs. placebo) and improved EF in females (56.7 +/- 3%, p < 0.05 vs. placebo) but not in males (50.6 +/- 3%, p = NS vs. placebo). Transcriptomic analysis using Rat_230-2.0 microarrays (Affymetrix) revealed that in females 19% of downregulated genes and 44% of upregulated genes post-MI were restored to normal by eplerenone. In contrast, eplerenone only restored 4% of overexpressed genes in males. Together, these data suggest that aldosterone blockade reduces MI-induced cardiac remodeling and phenotypic alterations of gene expression preferentially in females than in males. The use of transcriptomic signatures to detect greater benefit of eplerenone in females has potential implications for personalized medicine.