Rpl3l gene deletion in mice reduces heart weight over time.

Introduction: The ribosomal protein L3-like (RPL3L) is a heart and skeletal muscle-specific ribosomal protein and paralogue of the more ubiquitously expressed RPL3 protein. Mutations in the human RPL3L gene are linked to childhood cardiomyopathy and age-related atrial fibrillation, yet the function of RPL3L in the mammalian heart remains unknown. Methods and Results: Here, we observed that mouse cardiac ventricles express RPL3 at birth, where it is gradually replaced by RPL3L in adulthood but re-expressed with induction of hypertrophy in adults. Rpl3l gene-deleted mice were generated to examine the role of this gene in the heart, although Rpl3l -/- mice showed no overt changes in cardiac structure or function at baseline or after pressure overload hypertrophy, likely because RPL3 expression was upregulated and maintained in adulthood. mRNA expression analysis and ribosome profiling failed to show differences between the hearts of Rpl3l null and wild type mice in adulthood. Moreover, ribosomes lacking RPL3L showed no differences in localization within cardiomyocytes compared to wild type controls, nor was there an alteration in cardiac tissue ultrastructure or mitochondrial function in adult Rpl3l -/- mice. Similarly, overexpression of either RPL3 or RPL3L with adeno-associated virus -9 in the hearts of mice did not cause discernable pathology. However, by 18 months of age Rpl3l -/- null mice had significantly smaller hearts compared to wild type littermates. Conclusion: Thus, deletion of Rpl3l forces maintenance of RPL3 expression within the heart that appears to fully compensate for the loss of RPL3L, although older Rpl3l -/- mice showed a mild but significant reduction in heart weight.

An acute immune response underlies the benefit of cardiac stem cell therapy.

Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day1,2, despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biological effect3. The rationale for these cell therapy trials is derived from animal studies that show a modest but reproducible improvement in cardiac function in models of cardiac ischaemic injury4,5. Here we examine the mechanistic basis for cell therapy in mice after ischaemia-reperfusion injury, and find that-although heart function is enhanced-it is not associated with the production of new cardiomyocytes. Cell therapy improved heart function through an acute sterile immune response characterized by the temporal and regional induction of CCR2+ and CX3CR1+ macrophages. Intracardiac injection of two distinct types of adult stem cells, cells killed by freezing and thawing or a chemical inducer of the innate immune response all induced a similar regional accumulation of CCR2+ and CX3CR1+ macrophages, and provided functional rejuvenation to the heart after ischaemia-reperfusion injury. This selective macrophage response altered the activity of cardiac fibroblasts, reduced the extracellular matrix content in the border zone and enhanced the mechanical properties of the injured area. The functional benefit of cardiac cell therapy is thus due to an acute inflammatory-based wound-healing response that rejuvenates the infarcted area of the heart.

Inhibition of mitochondrial permeability transition by deletion of the ANT family and CypD.

The mitochondrial permeability transition pore (MPTP) has resisted molecular identification. The original model of the MPTP that proposed the adenine nucleotide translocator (ANT) as the inner membrane pore-forming component was challenged when mitochondria from Ant1/2 double null mouse liver still had MPTP activity. Because mice express three Ant genes, we reinvestigated whether the ANTs comprise the MPTP. Liver mitochondria from Ant1, Ant2, and Ant4 deficient mice were highly refractory to Ca2+-induced MPTP formation, and when also given cyclosporine A (CsA), the MPTP was completely inhibited. Moreover, liver mitochondria from mice with quadruple deletion of Ant1, Ant2, Ant4, and Ppif (cyclophilin D, target of CsA) lacked Ca2+-induced MPTP formation. Inner-membrane patch clamping in mitochondria from Ant1, Ant2, and Ant4 triple null mouse embryonic fibroblasts showed a loss of MPTP activity. Our findings suggest a model for the MPTP consisting of two distinct molecular components: The ANTs and an unknown species requiring CypD.

Inhibiting Fibronectin Attenuates Fibrosis and Improves Cardiac Function in a Model of Heart Failure.

BACKGROUND: Fibronectin (FN) polymerization is necessary for collagen matrix deposition and is a key contributor to increased abundance of cardiac myofibroblasts (MFs) after cardiac injury. We hypothesized that interfering with FN polymerization or its genetic ablation in fibroblasts would attenuate MF and fibrosis and improve cardiac function after ischemia/reperfusion (I/R) injury. METHODS: Mouse and human MFs were used to assess the impact of the FN polymerization inhibitor (pUR4) in attenuating pathological cellular features such as proliferation, migration, extracellular matrix deposition, and associated mechanisms. To evaluate the therapeutic potential of inhibiting FN polymerization in vivo, wild-type mice received daily intraperitoneal injections of either pUR4 or control peptide (III-11C) immediately after cardiac surgery for 7 consecutive days. Mice were analyzed 7 days after I/R to assess MF markers and inflammatory cell infiltration or 4 weeks after I/R to evaluate long-term effects of FN inhibition on cardiac function and fibrosis. Furthermore, inducible, fibroblast-restricted, FN gene-ablated (Tcf21MerCreMer; Fnflox) mice were used to evaluate cell specificity of FN expression and polymerization in the heart. RESULTS: pUR4 administration on activated MFs reduced FN and collagen deposition into the extracellular matrix and attenuated cell proliferation, likely mediated through decreased c-myc signaling. pUR4 also ameliorated fibroblast migration accompanied by increased ß1 integrin internalization and reduced levels of phosphorylated focal adhesion kinase protein. In vivo, daily administration of pUR4 for 7 days after I/R significantly reduced MF markers and neutrophil infiltration. This treatment regimen also significantly attenuated myocardial dysfunction, pathological cardiac remodeling, and fibrosis up to 4 weeks after I/R. Last, inducible ablation of FN in fibroblasts after I/R resulted in significant functional cardioprotection with reduced hypertrophy and fibrosis. The addition of pUR4 to the FN-ablated mice did not confer further cardioprotection, suggesting that the salutary effects of inhibiting FN polymerization may be mediated largely through effects on FN secreted from the cardiac fibroblast lineage. CONCLUSIONS: Inhibiting FN polymerization or cardiac fibroblast gene expression attenuates pathological properties of MFs in vitro and ameliorates adverse cardiac remodeling and fibrosis in an in vivo model of heart failure. Interfering with FN polymerization may be a new therapeutic strategy for treating cardiac fibrosis and heart failure.

Nemo-Like Kinase (NLK) Is a Pathological Signaling Effector in the Mouse Heart.

Nemo-like kinase (NLK) is an evolutionary conserved serine/threonine protein kinase implicated in development, proliferation and apoptosis regulation. Here we identified NLK as a gene product induced in the hearts of mice subjected to pressure overload or myocardial infarction injury, suggesting a potential regulatory role with pathological stimulation to this organ. To examine the potential functional consequences of increased NLK levels, cardiac-specific transgenic mice with inducible expression of this gene product were generated, as well as cardiac-specific Nlk gene-deleted mice. NLK transgenic mice demonstrated baseline cardiac hypertrophy, dilation, interstitial fibrosis, apoptosis and progression towards heart failure in response to two surgery-induced cardiac disease models. In contrast, cardiac-specific deletion of Nlk from the heart, achieved by crossing a Nlk-loxP allele containing mouse with either a mouse containing a ß-myosin heavy chain promoter driven Cre transgene or a tamoxifen inducible α-myosin heavy chain promoter containing transgene driving a MerCreMer cDNA, protected the mice from cardiac dysfunction following pathological stimuli. Mechanistically, NLK interacted with multiple proteins including the transcription factor Stat1, which was significantly increased in the hearts of NLK transgenic mice. These results indicate that NLK is a pathological effector in the heart.

Cathepsin S Contributes to the Pathogenesis of Muscular Dystrophy in Mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin protein compromises the stability of the sarcolemma membrane surrounding each muscle cell fiber, leading to membrane ruptures and leakiness that induces myofiber necrosis, a subsequent inflammatory response, and progressive tissue fibrosis with loss of functional capacity. Cathepsin S (Ctss) is a cysteine protease that is actively secreted in areas of tissue injury and ongoing inflammation, where it participates in extracellular matrix remodeling and healing. Here we show significant induction of Ctss expression and proteolytic activity following acute muscle injury or in muscle from mdx mice, a model of DMD. To examine the functional ramifications associated with greater Ctss expression, the Ctss gene was deleted in the mdx genetic background, resulting in protection from muscular dystrophy pathogenesis that included reduced myofiber turnover and histopathology, reduced fibrosis, and improved running capacity. Mechanistically, deletion of the Ctss gene in the mdx background significantly increased myofiber sarcolemmal membrane stability with greater expression and membrane localization of utrophin, integrins, and ß-dystroglycan, which anchor the membrane to the basal lamina and underlying cytoskeletal proteins. Consistent with these results, skeletal muscle-specific transgenic mice overexpressing Ctss showed increased myofiber necrosis, muscle histopathology, and a functional deficit reminiscent of muscular dystrophy. Hence, Ctss induction during muscular dystrophy is a pathologic event that partially underlies disease pathogenesis, and its inhibition might serve as a new therapeutic strategy in DMD.

Necroptosis Interfaces with MOMP and the MPTP in Mediating Cell Death.

During apoptosis the pro-death Bcl-2 family members Bax and Bak induce mitochondrial outer membrane permeabilization (MOMP) to mediate cell death. Recently, it was shown that Bax and Bak are also required for mitochondrial permeability transition pore (MPTP)-dependent necrosis, where, in their non-oligomeric state, they enhance permeability characteristics of the outer mitochondrial membrane. Necroptosis is another form of regulated necrosis involving the death receptors and receptor interacting protein kinases (RIP proteins, by Ripk genes). Here, we show cells or mice deficient for Bax/Bak or cyclophilin D, a protein that regulates MPTP opening, are resistant to cell death induced by necroptotic mediators. We show that Bax/Bak oligomerization is required for necroptotic cell death and that this oligomerization reinforces MPTP opening. Mechanistically, we observe mixed lineage kinase domain-like (MLKL) protein and cofilin-1 translocation to the mitochondria following necroptosis induction, while expression of the mitochondrial matrix isoform of the antiapoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), is significantly reduced. Some of these effects are lost with necroptosis inhibition in Bax/Bak1 double null, Ppif-/-, or Ripk3-/- fibroblasts. Hence, downstream mechanisms of cell death induced by necroptotic stimuli utilize both Bax/Bak to generate apoptotic pores in the outer mitochondrial membrane as well as MPTP opening in association with known mitochondrial death modifying proteins.

The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart.

In the heart, augmented Ca(2+) fluxing drives contractility and ATP generation through mitochondrial Ca(2+) loading. Pathologic mitochondrial Ca(2+) overload with ischemic injury triggers mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte death. Mitochondrial Ca(2+) uptake is primarily mediated by the mitochondrial Ca(2+) uniporter (MCU). Here, we generated mice with adult and cardiomyocyte-specific deletion of Mcu, which produced mitochondria refractory to acute Ca(2+) uptake, with impaired ATP production, and inhibited MPTP opening upon acute Ca(2+) challenge. Mice lacking Mcu in the adult heart were also protected from acute ischemia-reperfusion injury. However, resting/basal mitochondrial Ca(2+) levels were normal in hearts of Mcu-deleted mice, and mitochondria lacking MCU eventually loaded with Ca(2+) after stress stimulation. Indeed, Mcu-deleted mice were unable to immediately sprint on a treadmill unless warmed up for 30 min. Hence, MCU is a dedicated regulator of short-term mitochondrial Ca(2+) loading underlying a "fight-or-flight" response that acutely matches cardiac workload with ATP production.

Na+ dysregulation coupled with Ca2+ entry through NCX1 promotes muscular dystrophy in mice.

Unregulated Ca(2+) entry is thought to underlie muscular dystrophy. Here, we generated skeletal-muscle-specific transgenic (TG) mice expressing the Na(+)-Ca(2+) exchanger 1 (NCX1) to model its identified augmentation during muscular dystrophy. The NCX1 transgene induced dystrophy-like disease in all hind-limb musculature, as well as exacerbated the muscle disease phenotypes in δ-sarcoglycan (Sgcd(-/-)), Dysf(-/-), and mdx mouse models of muscular dystrophy. Antithetically, muscle-specific deletion of the Slc8a1 (NCX1) gene diminished hind-limb pathology in Sgcd(-/-) mice. Measured increases in baseline Na(+) and Ca(2+) in dystrophic muscle fibers of the hind-limb musculature predicts a net Ca(2+) influx state due to reverse-mode operation of NCX1, which mediates disease. However, the opposite effect is observed in the diaphragm, where NCX1 overexpression mildly protects from dystrophic disease through a predicted enhancement in forward-mode NCX1 operation that reduces Ca(2+) levels. Indeed, Atp1a2(+/-) (encoding Na(+)-K(+) ATPase α2) mice, which have reduced Na(+) clearance rates that would favor NCX1 reverse-mode operation, showed exacerbated disease in the hind limbs of NCX1 TG mice, similar to treatment with the Na(+)-K(+) ATPase inhibitor digoxin. Treatment of Sgcd(-/-) mice with ranolazine, a broadly acting Na(+) channel inhibitor that should increase NCX1 forward-mode operation, reduced muscular pathology.

Enhanced Ca²âº influx from STIM1-Orai1 induces muscle pathology in mouse models of muscular dystrophy.

Muscular dystrophy is a progressive muscle wasting disease that is thought to be initiated by unregulated Ca(2+) influx into myofibers leading to their death. Store-operated Ca(2+) entry (SOCE) through sarcolemmal Ca(2+) selective Orai1 channels in complex with STIM1 in the sarcoplasmic reticulum is one such potential disease mechanism for pathologic Ca(2+) entry. Here, we generated a mouse model of STIM1 overexpression in skeletal muscle to determine whether this type of Ca(2+) entry could induce muscular dystrophy. Myofibers from muscle-specific STIM1 transgenic mice showed a significant increase in SOCE in skeletal muscle, modeling an observed increase in the same current in dystrophic myofibers. Histological and biochemical analysis of STIM1 transgenic mice showed fulminant muscle disease characterized by myofiber necrosis, swollen mitochondria, infiltration of inflammatory cells, enhanced interstitial fibrosis and elevated serum creatine kinase levels. This dystrophic-like disease in STIM1 transgenic mice was abrogated by crossing in a transgene expressing a dominant-negative Orai1 (dnOrai1) mutant. The dnOrai1 transgene also significantly reduced the severity of muscular dystrophy in both mdx (dystrophin mutant mice) and δ-sarcoglycan-deficient (Sgcd(-/-)) mouse models of disease. Hence, Ca(2+) influx across an unstable sarcolemma due to increased activity of a STIM1-Orai1 complex is a disease determinant in muscular dystrophy, and hence, SOCE represents a potential therapeutic target.

Mitigation of muscular dystrophy in mice by SERCA overexpression in skeletal muscle.

Muscular dystrophies (MDs) comprise a group of degenerative muscle disorders characterized by progressive muscle wasting and often premature death. The primary defect common to most MDs involves disruption of the dystrophin-glycoprotein complex (DGC). This leads to sarcolemmal instability and Ca(2+) influx, inducing cellular necrosis. Here we have shown that the dystrophic phenotype observed in δ-sarcoglycan­null (Sgcd(­/­)) mice and dystrophin mutant mdx mice is dramatically improved by skeletal muscle­specific overexpression of sarcoplasmic reticulum Ca(2+) ATPase 1 (SERCA1). Rates of myofiber central nucleation, tissue fibrosis, and serum creatine kinase levels were dramatically reduced in Sgcd(­/­) and mdx mice with the SERCA1 transgene, which also rescued the loss of exercise capacity in Sgcd(­/­) mice. Adeno-associated virus­SERCA2a (AAV-SERCA2a) gene therapy in the gastrocnemius muscle of Sgcd(­/­) mice mitigated dystrophic disease. SERCA1 overexpression reversed a defect in sarcoplasmic reticulum Ca(2+) reuptake that characterizes dystrophic myofibers and reduced total cytosolic Ca(2+). Further, SERCA1 overexpression almost completely rescued the dystrophic phenotype in a mouse model of MD driven solely by Ca(2+) influx. Mitochondria isolated from the muscle of SERCA1-Sgcd(­/­) mice were no longer swollen and calpain activation was reduced, suggesting protection from Ca(2+)-driven necrosis. Our results suggest a novel therapeutic approach using SERCA1 to abrogate the altered intracellular Ca(2+) levels that underlie most forms of MD.

Genetic manipulation of periostin expression in the heart does not affect myocyte content, cell cycle activity, or cardiac repair.

Following a pathological insult, the adult mammalian heart undergoes hypertrophic growth and remodeling of the extracellular matrix. Although a small subpopulation of cardiomyocytes can reenter the cell cycle following cardiac injury, the myocardium is largely thought to be incapable of significant regeneration. Periostin, an extracellular matrix protein, has recently been proposed to induce reentry of differentiated cardiomyocytes back into the cell cycle and promote meaningful repair following myocardial infarction. Here, we show that although periostin is induced in the heart following injury, it does not stimulate DNA synthesis, mitosis, or cytokinesis of cardiomyocytes in vitro or in vivo. Mice lacking the gene encoding periostin and mice with inducible overexpression of full-length periostin were analyzed at baseline and after myocardial infarction. There was no difference in heart size or a change in cardiomyocyte number in either periostin transgenic or gene-targeted mice at baseline. Quantification of proliferating myocytes in the periinfarct area showed no difference between periostin-overexpressing and -null mice compared with strain-matched controls. In support of these observations, neither overexpression of periostin in cell culture, via an adenoviral vector, nor stimulation with recombinant protein induced DNA synthesis, mitosis, or cytokinesis. Periostin is a regulator of cardiac remodeling and hypertrophy and may be a reasonable pharmacological target to mitigate heart failure, but manipulation of this protein appears to have no obvious effect on myocardial regeneration.

Cardiomyocyte GATA4 functions as a stress-responsive regulator of angiogenesis in the murine heart.

The transcription factor GATA4 is a critical regulator of cardiac gene expression, modulating cardiomyocyte differentiation and adaptive responses of the adult heart. We report what we believe to be a novel function for GATA4 in murine cardiomyocytes as a nodal regulator of cardiac angiogenesis. Conditional overexpression of GATA4 within adult cardiomyocytes increased myocardial capillary and small conducting vessel densities and increased coronary flow reserve and perfusion-dependent cardiac contractility. Coculture of HUVECs with either GATA4-expressing cardiomyocytes or with myocytes expressing a dominant-negative form of GATA4 enhanced or reduced HUVEC tube formation, respectively. Expression of GATA4 in skeletal muscle by adenoviral gene transfer enhanced capillary densities and hindlimb perfusion following femoral artery ablation. Deletion of Gata4 specifically from cardiomyocytes reduced myocardial capillary density and prevented pressure overload-augmented angiogenesis in vivo. GATA4 induced the angiogenic factor VEGF-A, directly binding the Vegf-A promoter and enhancing transcription. GATA4-overexpressing mice showed increased levels of cardiac VEGF-A, while Gata4-deleted mice demonstrated decreased VEGF-A levels. The induction of HUVEC tube formation in GATA4-overexpressing cocultured myocytes was blocked with a VEGF receptor antagonist. Pressure overload-induced dysfunction in Gata4-deleted hearts was partially rescued by adenoviral gene delivery of VEGF and angiopoietin-1. To our knowledge, these results demonstrate [corrected] a previously unrecognized function for GATA4 as a regulator of cardiac angiogenesis through a nonhypoxic, load, and/or disease-responsive mechanism.

Age-dependent effect of myostatin blockade on disease severity in a murine model of limb-girdle muscular dystrophy.

Myostatin (MSTN) is a muscle-specific secreted peptide that functions to limit muscle growth through an autocrine regulatory feedback loop. Loss of MSTN activity in cattle, mice, and humans leads to a profound phenotype of muscle overgrowth, associated with more and larger fibers and enhanced regenerative capacity. Deletion of MSTN in the mdx mouse model of Duchenne muscular dystrophy enhances muscle mass and reduces disease severity. In contrast, loss of MSTN activity in the dyW/dyW mouse model of laminin-deficient congenital muscular dystrophy, a much more severe and lethal disease model, does not improve all aspects of muscle pathology. Here we examined disease severity associated with myostatin (mstn-/-) deletion in mice nullizygous for delta-sarcoglycan (scgd-/-), a model of limb-girdle muscular dystrophy. Early loss of MSTN activity achieved either by monoclonal antibody administration or by gene deletion each improved muscle mass, regeneration, and reduced fibrosis in scgd-/- mice. However, antibody-mediated inhibition of MSTN in late-stage dystrophic scgd-/- mice did not improve disease. These findings suggest that MSTN inhibition may benefit muscular dystrophy when instituted early or if disease is relatively mild but that MSTN inhibition in severely affected or late-stage disease may be ineffective.