Anchored p90 ribosomal S6 kinase 3 is required for cardiac myocyte hypertrophy.

RATIONALE: Cardiac myocyte hypertrophy is the main compensatory response to chronic stress on the heart. p90 ribosomal S6 kinase (RSK) family members are effectors for extracellular signal-regulated kinases that induce myocyte growth. Although increased RSK activity has been observed in stressed myocytes, the functions of individual RSK family members have remained poorly defined, despite being potential therapeutic targets for cardiac disease. OBJECTIVE: To demonstrate that type 3 RSK (RSK3) is required for cardiac myocyte hypertrophy. METHODS AND RESULTS: RSK3 contains a unique N-terminal domain that is not conserved in other RSK family members. We show that this domain mediates the regulated binding of RSK3 to the muscle A-kinase anchoring protein scaffold, defining a novel kinase anchoring event. Disruption of both RSK3 expression using RNA interference and RSK3 anchoring using a competing muscle A-kinase anchoring protein peptide inhibited the hypertrophy of cultured myocytes. In vivo, RSK3 gene deletion in the mouse attenuated the concentric myocyte hypertrophy induced by pressure overload and catecholamine infusion. CONCLUSIONS: Taken together, these data demonstrate that anchored RSK3 transduces signals that modulate pathologic myocyte growth. Targeting of signaling complexes that contain select kinase isoforms should provide an approach for the specific inhibition of cardiac myocyte hypertrophy and for the development of novel strategies for the prevention and treatment of heart failure.

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.

Transendocardial mesenchymal stem cells and mononuclear bone marrow cells for ischemic cardiomyopathy: the TAC-HFT randomized trial.

IMPORTANCE: Whether culture-expanded mesenchymal stem cells or whole bone marrow mononuclear cells are safe and effective in chronic ischemic cardiomyopathy is controversial. OBJECTIVE: To demonstrate the safety of transendocardial stem cell injection with autologous mesenchymal stem cells (MSCs) and bone marrow mononuclear cells (BMCs) in patients with ischemic cardiomyopathy. DESIGN, SETTING, AND PATIENTS: A phase 1 and 2 randomized, blinded, placebo-controlled study involving 65 patients with ischemic cardiomyopathy and left ventricular (LV) ejection fraction less than 50% (September 1, 2009-July 12, 2013). The study compared injection of MSCs (n=19) with placebo (n = 11) and BMCs (n = 19) with placebo (n = 10), with 1 year of follow-up. INTERVENTIONS: Injections in 10 LV sites with an infusion catheter. MAIN OUTCOMES AND MEASURES: Treatment-emergent 30-day serious adverse event rate defined as a composite of death, myocardial infarction, stroke, hospitalization for worsening heart failure, perforation, tamponade, or sustained ventricular arrhythmias. RESULTS: No patient had a treatment-emergent serious adverse events at day 30. The 1-year incidence of serious adverse events was 31.6% (95% CI, 12.6% to 56.6%) for MSCs, 31.6% (95% CI, 12.6%-56.6%) for BMCs, and 38.1% (95% CI, 18.1%-61.6%) for placebo. Over 1 year, the Minnesota Living With Heart Failure score improved with MSCs (-6.3; 95% CI, -15.0 to 2.4; repeated measures of variance, P=.02) and with BMCs (-8.2; 95% CI, -17.4 to 0.97; P=.005) but not with placebo (0.4; 95% CI, -9.45 to 10.25; P=.38). The 6-minute walk distance increased with MSCs only (repeated measures model, P = .03). Infarct size as a percentage of LV mass was reduced by MSCs (-18.9%; 95% CI, -30.4 to -7.4; within-group, P = .004) but not by BMCs (-7.0%; 95% CI, -15.7% to 1.7%; within-group, P = .11) or placebo (-5.2%; 95% CI, -16.8% to 6.5%; within-group, P = .36). Regional myocardial function as peak Eulerian circumferential strain at the site of injection improved with MSCs (-4.9; 95% CI, -13.3 to 3.5; within-group repeated measures, P = .03) but not BMCs (-2.1; 95% CI, -5.5 to 1.3; P = .21) or placebo (-0.03; 95% CI, -1.9 to 1.9; P = .14). Left ventricular chamber volume and ejection fraction did not change. CONCLUSIONS AND RELEVANCE: Transendocardial stem cell injection with MSCs or BMCs appeared to be safe for patients with chronic ischemic cardiomyopathy and LV dysfunction. Although the sample size and multiple comparisons preclude a definitive statement about safety and clinical effect, these results provide the basis for larger studies to provide definitive evidence about safety and to assess efficacy of this new therapeutic approach. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00768066.

Bone marrow mesenchymal stem cells stimulate cardiac stem cell proliferation and differentiation.

RATIONALE: The regenerative potential of the heart is insufficient to fully restore functioning myocardium after injury, motivating the quest for a cell-based replacement strategy. Bone marrow-derived mesenchymal stem cells (MSCs) have the capacity for cardiac repair that appears to exceed their capacity for differentiation into cardiac myocytes. OBJECTIVE: Here, we test the hypothesis that bone marrow derived MSCs stimulate the proliferation and differentiation of endogenous cardiac stem cells (CSCs) as part of their regenerative repertoire. METHODS AND RESULTS: Female Yorkshire pigs (n=31) underwent experimental myocardial infarction (MI), and 3 days later, received transendocardial injections of allogeneic male bone marrow-derived MSCs, MSC concentrated conditioned medium (CCM), or placebo (Plasmalyte). A no-injection control group was also studied. MSCs engrafted and differentiated into cardiomyocytes and vascular structures. In addition, endogenous c-kit(+) CSCs increased 20-fold in MSC-treated animals versus controls (P<0.001), there was a 6-fold increase in GATA-4(+) CSCs in MSC versus control (P<0.001), and mitotic myocytes increased 4-fold (P=0.005). Porcine endomyocardial biopsies were harvested and plated as organotypic cultures in the presence or absence of MSC feeder layers. In vitro, MSCs stimulated c-kit(+) CSCs proliferation into enriched populations of adult cardioblasts that expressed Nkx2-5 and troponin I. CONCLUSIONS: MSCs stimulate host CSCs, a new mechanism of action underlying successful cell-based therapeutics.

Allogeneic mesenchymal stem cells restore cardiac function in chronic ischemic cardiomyopathy via trilineage differentiating capacity.

The mechanism(s) underlying cardiac reparative effects of bone marrow-derived mesenchymal stem cells (MSC) remain highly controversial. Here we tested the hypothesis that MSCs regenerate chronically infarcted myocardium through mechanisms comprising long-term engraftment and trilineage differentiation. Twelve weeks after myocardial infarction, female swine received catheter-based transendocardial injections of either placebo (n = 4) or male allogeneic MSCs (200 million; n = 6). Animals underwent serial cardiac magnetic resonance imaging, and in vivo cell fate was determined by co-localization of Y-chromosome (Y(pos)) cells with markers of cardiac, vascular muscle, and endothelial lineages. MSCs engrafted in infarct and border zones and differentiated into cardiomyocytes as ascertained by co-localization with GATA-4, Nkx2.5, and alpha-sarcomeric actin. In addition, Y(pos) MSCs exhibited vascular smooth muscle and endothelial cell differentiation, contributing to large and small vessel formation. Infarct size was reduced from 19.3 +/- 1.7% to 13.9 +/- 2.0% (P < 0.001), and ejection fraction (EF) increased from 35.0 +/- 1.7% to 41.3 +/- 2.7% (P < 0.05) in MSC but not placebo pigs over 12 weeks. This was accompanied by increases in regional contractility and myocardial blood flow (MBF), particularly in the infarct border zone. Importantly, MSC engraftment correlated with functional recovery in contractility (R = 0.85, P < 0.05) and MBF (R = 0.76, P < 0.01). Together these findings demonstrate long-term MSC survival, engraftment, and trilineage differentiation following transplantation into chronically scarred myocardium. MSCs are an adult stem cell with the capacity for cardiomyogenesis and vasculogenesis which contribute, at least in part, to their ability to repair chronically scarred myocardium.

Rationale and design of the Transendocardial Injection of Autologous Human Cells (bone marrow or mesenchymal) in Chronic Ischemic Left Ventricular Dysfunction and Heart Failure Secondary to Myocardial Infarction (TAC-HFT) trial: A randomized, double-blind, placebo-controlled study of safety and efficacy.

Although there is tremendous interest in stem cell (SC)-based therapies for cardiomyopathy caused by chronic myocardial infarction, many unanswered questions regarding the best approach remain. The TAC-HFT study is a phase I/II randomized, double-blind, placebo-controlled trial designed to address several of these questions, including the optimal cell type, delivery technique, and population. This trial compares autologous mesenchymal SCs (MSCs) and whole bone marrow mononuclear cells (BMCs). In addition, the study will use a novel helical catheter to deliver cells transendocardially. Although most trials have used intracoronary delivery, the optimal method is unknown and data suggest that the transendocardial approach may have important advantages. Several trials support the benefit of SCs in patients with chronic ischemic cardiomyopathy (ICMP), although the sample sizes have been small and the number of trials sparse. After a pilot phase of 8 patients, 60 patients with ICMP (left ventricular ejection fraction 15%-50%) will be randomized to group A (30 patients further randomized to receive MSC injection or placebo in a 2:1 fashion) or group B (30 patients further randomized to BMCs or placebo in a 2:1 fashion). All patients will undergo bone marrow aspiration and transendocardial injection of SCs or placebo. The primary and secondary objectives are, respectively, to demonstrate the safety and efficacy (determined primarily by cardiac magnetic resonance imaging) of BMCs and MSCs administered transendocardially in patients with ICMP.

Cardiac nitric oxide synthase-1 localization within the cardiomyocyte is accompanied by the adaptor protein, CAPON.

The mechanism(s) regulating nitric oxide synthase-1 (NOS1) localization within the cardiac myocyte in health and disease remains unknown. Here we tested the hypothesis that the PDZ-binding domain interaction between CAPON (carboxy-terminal PDZ ligand of NOS1), a NOS1 adaptor protein and NOS1, contribute to NOS1 localization in specific organelles within cardiomyocytes. Ventricular cardiomyocytes and whole heart homogenates were isolated from sham and post-myocardial infarction (MI) wild-type (C57BL/6) and NOS1(-/-) female mice for quantification of CAPON protein expression levels. NOS1, CAPON, xanthine oxidoreductase and Dexras1, a CAPON binding partner, were all present and enriched in isolated cardiac sarcoplasmic reticulum (SR) fractions. CAPON co-immunoprecipitated with the mu and alpha isoforms of NOS1 in whole heart lysates, and co-localization of CAPON and NOS1 was demonstrated in the SR and mitochondria with dual immuno-gold electron microscopy. Following MI, CAPON and NOS1 both redistributed to caveolae and colocalized with caveolin-3. In addition, following MI, expression level of CAPON remained unchanged and Dexras1 was reduced, CAPON binding to xanthine oxidoreductase was augmented and the plasma membrane calcium ATPase (PMCA) increased. In NOS1 deficient myocytes, CAPON abundance in the SR was reduced, and redistribution to caveolae and PMCA binding after MI was absent. Together these findings support the hypothesis that NOS1 redistribution in injured myocardium requires the formation of a complex with the PDZ adaptor protein CAPON.

Increased potency of cardiac stem cells compared with bone marrow mesenchymal stem cells in cardiac repair.

Whereas cardiac-derived c-kit(+) stem cells (CSCs) and bone marrow-derived mesenchymal stem cells (MSCs) are undergoing clinical trials testing safety and efficacy as a cell-based therapy, the relative therapeutic and biologic efficacy of these two cell types is unknown. We hypothesized that human CSCs have greater ability than MSCs to engraft, differentiate, and improve cardiac function. We compared intramyocardial injection of human fetal CSCs (36,000) with two doses of adult MSCs (36,000 and 1,000,000) or control (phosphate buffered saline) in nonobese diabetic/severe combined immune deficiency mice after coronary artery ligation. The myocardial infarction-induced enlargement in left ventricular chamber dimensions was ameliorated by CSCs (p < .05 for diastolic and systolic volumes), as was the decline in ejection fraction (EF; p < .05). Whereas 1 × 10(6) MSCs partially ameliorated ventricular remodeling and improved EF to a similar degree as CSCs, 36,000 MSCs did not influence chamber architecture or function. All cell therapies improved myocardial contractility, but CSCs preferentially reduced scar size and reduced vascular afterload. Engraftment and trilineage differentiation was substantially greater with CSCs than with MSCs. Adult-cultured c-kit(+)CSCs were less effective than fetal, but were still more potent than high-dose MSCs. These data demonstrate enhanced CSC engraftment, differentiation, and improved cardiac remodeling and function in ischemic heart failure. MSCs required a 30-fold greater dose than CSCs to improve cardiac function and anatomy. Together, these findings demonstrate a greater potency of CSCs than bone marrow MSCs in cardiac repair.

Absence of myocardial thyroid hormone inactivating deiodinase results in restrictive cardiomyopathy in mice.

Cardiac injury induces myocardial expression of the thyroid hormone inactivating type 3 deiodinase (D3), which in turn dampens local thyroid hormone signaling. Here, we show that the D3 gene (Dio3) is a tissue-specific imprinted gene in the heart, and thus, heterozygous D3 knockout (HtzD3KO) mice constitute a model of cardiac D3 inactivation in an otherwise systemically euthyroid animal. HtzD3KO newborns have normal hearts but later develop restrictive cardiomyopathy due to cardiac-specific increase in thyroid hormone signaling, including myocardial fibrosis, impaired myocardial contractility, and diastolic dysfunction. In wild-type littermates, treatment with isoproterenol-induced myocardial D3 activity and an increase in the left ventricular volumes, typical of cardiac remodeling and dilatation. Remarkably, isoproterenol-treated HtzD3KO mice experienced a further decrease in left ventricular volumes with worsening of the diastolic dysfunction and the restrictive cardiomyopathy, resulting in congestive heart failure and increased mortality. These findings reveal crucial roles for Dio3 in heart function and remodeling, which may have pathophysiologic implications for human restrictive cardiomyopathy.

FGF23 induces left ventricular hypertrophy.

Chronic kidney disease (CKD) is a public health epidemic that increases risk of death due to cardiovascular disease. Left ventricular hypertrophy (LVH) is an important mechanism of cardiovascular disease in individuals with CKD. Elevated levels of FGF23 have been linked to greater risks of LVH and mortality in patients with CKD, but whether these risks represent causal effects of FGF23 is unknown. Here, we report that elevated FGF23 levels are independently associated with LVH in a large, racially diverse CKD cohort. FGF23 caused pathological hypertrophy of isolated rat cardiomyocytes via FGF receptor-dependent activation of the calcineurin-NFAT signaling pathway, but this effect was independent of klotho, the coreceptor for FGF23 in the kidney and parathyroid glands. Intramyocardial or intravenous injection of FGF23 in wild-type mice resulted in LVH, and klotho-deficient mice demonstrated elevated FGF23 levels and LVH. In an established animal model of CKD, treatment with an FGF-receptor blocker attenuated LVH, although no change in blood pressure was observed. These results unveil a klotho-independent, causal role for FGF23 in the pathogenesis of LVH and suggest that chronically elevated FGF23 levels contribute directly to high rates of LVH and mortality in individuals with CKD.

Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts.

Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

Early improvement in cardiac tissue perfusion due to mesenchymal stem cells.

The underlying mechanism(s) of improved left ventricular function (LV) due to mesenchymal stem cell (MSC) administration after myocardial infarction (MI) remains highly controversial. Myocardial regeneration and neovascularization, which leads to increased tissue perfusion, are proposed mechanisms. Here we demonstrate that delivery of MSCs 3 days after MI increased tissue perfusion in a manner that preceded improved LV function in a porcine model. MI was induced in pigs by 60-min occlusion of the left anterior descending coronary artery, followed by reperfusion. Pigs were assigned to receive intramyocardial injection of allogeneic MSCs (200 million, approximately 15 injections) (n = 10), placebo (n = 6), or no intervention (n = 8). Resting myocardial blood flow (MBF) was serially assessed by first-pass perfusion magnetic resonance imaging (MRI) over an 8-wk period. Over the first week, resting MBF in the infarct area of MSC-treated pigs increased compared with placebo-injected and untreated animals [0.17 +/- 0.03, 0.09 +/- 0.01, and 0.08 +/- 0.01, respectively, signal intensity ratio of MI to left ventricular blood pool (LVBP); P < 0.01 vs. placebo, P < 0.01 vs. nontreated]. In contrast, the signal intensity ratios of the three groups were indistinguishable at weeks 4 and 8. However, MSC-treated animals showed larger, more mature vessels and less apoptosis in the infarct zones and improved regional and global LV function at week 8. Together these findings suggest that an early increase in tissue perfusion precedes improvements in LV function and a reduction in apoptosis in MSC-treated hearts. Cardiac MRI-based measures of blood flow may be a useful tool to predict a successful myocardial regenerative process after MSC treatment.

Multimodality noninvasive imaging demonstrates in vivo cardiac regeneration after mesenchymal stem cell therapy.

OBJECTIVES: The purpose of this study was to test the hypothesis, with noninvasive multimodality imaging, that allogeneic mesenchymal stem cells (MSCs) produce and/or stimulate active cardiac regeneration in vivo after myocardial infarction (MI). BACKGROUND: Although intramyocardial injection of allogeneic MSCs improves global cardiac function after MI, the mechanism(s) underlying this phenomenon are incompletely understood. METHODS: We employed magnetic resonance imaging (MRI) and multi-detector computed tomography (MDCT) imaging in MSC-treated pigs (n = 10) and control subjects (n = 12) serially for a 2-month period after anterior MI. A sub-endocardial rim of tissue, demonstrated with MDCT, was assessed for regional contraction with MRI tagging. Rim thickness was also measured on gross pathological specimens, to confirm the findings of the MDCT imaging, and the size of cardiomyocytes was measured in the sub-endocardial rim and the non-infarct zone. RESULTS: Multi-detector computed tomography demonstrated increasing thickness of sub-endocardial viable myocardium in the infarct zone in MSC-treated animals (1.0 +/- 0.2 mm to 2.0 +/- 0.3 mm, 1 and 8 weeks after MI, respectively, p = 0.028, n = 4) and a corresponding reduction in infarct scar (5.1 +/- 0.5 mm to 3.6 +/- 0.2 mm, p = 0.044). No changes occurred in control subjects (n = 4). Tagging MRI demonstrated time-dependent recovery of active contractility paralleling new tissue appearance. This rim was composed of morphologically normal cardiomyocytes, which were smaller in MSC-treated versus control subjects (11.6 +/- 0.2 mum vs. 12.6 +/- 0.2 mum, p < 0.05). CONCLUSIONS: With serially obtained MRI and MDCT, we demonstrate in vivo reappearance of myocardial tissue in the MI zone accompanied by time-dependent restoration of contractile function. These data are consistent with a regenerative process, highlight the value of noninvasive multimodality imaging to assess the structural and functional basis for myocardial regenerative strategies, and have potential clinical applications.