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Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day.sup.1,2, despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biological effect.sup.3. 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 injury.sup.4,5. 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.sup.+ and CX3CR1.sup.+ 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.sup.+ and CX3CR1.sup.+ 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. Cardiac stem cell therapy in mouse models of ischaemia-reperfusion injury demonstrates that improvement in heart function is linked to an immune response characterized by the induction of CCR2.sup.+ and CX3CR1.sup.+ macrophages., Author(s): Ronald J. Vagnozzi [sup.1] , Marjorie Maillet [sup.1] , Michelle A. Sargent [sup.1] , Hadi Khalil [sup.1] , Anne Katrine Z. Johansen [sup.1] , Jennifer A. Schwanekamp [sup.2] , [...]

Fibroblasts are a dynamic cell type that achieve selective differentiated states to mediate acute wound healing and long-term tissue remodeling with scarring. With myocardial infarction injury, cardiomyocytes are replaced by secreted extracellular matrix proteins produced by proliferating and differentiating fibroblasts. Here, we employed 3 different mouse lineage tracing models and stage-specific gene profiling to phenotypically analyze and classify resident cardiac fibroblast dynamics during myocardial infarction injury and stable scar formation. Fibroblasts were activated and highly proliferative, reaching a maximum rate within 2 to 4 days after infarction injury, at which point they expanded 3.5-fold and were maintained long term. By 3 to 7 days, these cells differentiated into myofibroblasts that secreted abundant extracellular matrix proteins and expressed smooth muscle [alpha]-actin to structurally support the necrotic area. By 7 to 10 days, myofibroblasts lost proliferative ability and smooth muscle [alpha]-actin expression as the collagen-containing extracellular matrix and scar fully matured. However, these same lineage-traced initial fibroblasts persisted within the scar, achieving a new molecular and stable differentiated state referred to as a matrifibrocyte, which was also observed in the scars of human hearts. These cells express common and unique extracellular matrix and tendon genes that are more specialized to support the mature scar., Introduction Fibroblasts are a unique cell type of mesenchymal origin that are present in essentially all tissues and organs, where they regulate extracellular matrix (ECM) production and acute wound healing [...]

Rationale: The adult cardiac extracellular matrix (ECM) is largely comprised of type I collagen. In addition to serving as the primary structural support component of the cardiac ECM, type I collagen also provides an organizational platform for other ECM proteins, matricellular proteins, and signaling components that impact cellular stress sensing in vivo. Objective: Here we investigated how the content and integrity of type I collagen affect cardiac structure function and response to injury. Methods and Results: We generated and characterized Col1a2−/− mice using standard gene targeting. Col1a2−/− mice were viable, although by young adulthood their hearts showed alterations in ECM mechanical properties, as well as an unanticipated activation of cardiac fibroblasts and induction of a progressive fibrotic response. This included augmented TGFβ activity, increases in fibroblast number, and progressive cardiac hypertrophy, with reduced functional performance by 9 months of age. Col1a2-loxP-targeted mice were also generated and crossed with the tamoxifen-inducible Postn-MerCreMer mice to delete the Col1a2 gene in myofibroblasts with pressure overload injury. Interestingly, while germline Col1a2−/− mice showed gradual pathologic hypertrophy and fibrosis with aging, the acute deletion of Col1a2 from activated adult myofibroblasts showed a loss of total collagen deposition with acute cardiac injury and an acute reduction in pressure overload-induce cardiac hypertrophy. However, this reduction in hypertrophy due to myofibroblast-specific Col1a2 deletion was lost after 2 and 6 weeks of pressure overload, as fibrotic deposition accumulated. Conclusions: Defective type I collagen in the heart alters the structural integrity of the ECM and leads to cardiomyopathy in adulthood, with fibroblast expansion, activation, and alternate fibrotic ECM deposition. However, acute inhibition of type I collagen production can have an anti-fibrotic and anti-hypertrophic effect. [ABSTRACT FROM AUTHOR]

To better understand the genetic basis of heart disease, we identified a variant in the Flightless-I homolog (FLII) gene that generates a R1243H missense change and predis-poses to cardiac remodeling across multiple previous human genome-wide association studies (GWAS). Since this gene is of unknown function in the mammalian heart we generated gain- and loss-of-function genetically altered mice, as well as knock-in mice with the syntenic R1245H amino acid substitution, which showed that Flii protein binds the sarcomeric actin thin filament and influences its length. Deletion of Flii from the heart, or mice with the R1245H amino acid substitution, show cardiomyopathy due to shortening of the actin thin filaments. Mechanistically, Flii is a known actin binding protein that we show associates with tropomodulin-1 (TMOD1) to regulate sarcomere thin filament length. Indeed, overexpression of leiomodin-2 in the heart, which lengthens the actin-containing thin filaments, partially rescued disease due to heart-specific deletion of Flii. Collectively, the identified FLII human variant likely increases cardiomyopathy risk through an alteration in sarcomere structure and associated contractile dynamics, like other sarcomere gene-based familial cardiomyopathies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Antagonists of L-type [Ca.sup.2+] channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1[C.sup.+/-] mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1[C.sup.-/+] mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1[C.sup.+/-] mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1[C.sup.+/-] controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocytespecific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanisti-cally, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum [Ca.sup.2+] release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease., Introduction Voltage-gated L-type [Ca.sup.2+] channels (LTCCs) are the primary source of [Ca.sup.2+] influx to initiate cardiac excitation-contraction coupling (ECC) (1, 2). The molecular composition of the LTCC in cardiomyocytes includes [...]

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.sup.2+] influx, inducing cellular necrosis. Here we have shown that the dystrophic phenotype observed in Δ-sarcoglycan-null ([Sgcd.sup.-/-]) mice and dystrophin mutant mdx mice is dramatically improved by skeletal muscle-specific overexpression of sarcoplasmic reticulum [Ca.sup.2+] ATPase 1 (SERCA1). Rates of myofiber central nucleation, tissue fibrosis, and serum creatine kinase levels were dramatically reduced in [Sgcd.sup.-/-] and mdx mice with the SERCA1 transgene, which also rescued the loss of exercise capacity in [Sgcd.sup.-/-] mice. Adeno-associated virus-SERCA2a (AAV-SERCA2a) gene therapy in the gastrocnemius muscle of [Sgcd.sup.-/-] mice mitigated dystrophic disease. SERCA1 overexpression reversed a defect in sarcoplasmic reticulum [Ca.sup.2+] reuptake that characterizes dystrophic myofibers and reduced total cytosolic [Ca.sup.2+]. Further, SERCA1 overexpression almost completely rescued the dystrophic phenotype in a mouse model of MD driven solely by [Ca.sup.2+] influx. Mitochondria isolated from the muscle of SERCA1-[Sgcd.sup.-/-] mice were no longer swollen and calpain activation was reduced, suggesting protection from [Ca.sup.2+]-driven necrosis. Our results suggest a novel therapeutic approach using SERCA1 to abrogate the altered intracellular [Ca.sup.2+] levels that underlie most forms of MD., Introduction MD broadly encompasses a diverse group of genetic disorders that result in progressive muscle wasting and premature death (1). The majority of genes identified in patients with MD appear [...]

How [Ca.sup.2+]-dependent signaling effectors are regulated in cardiomyocytes, given the extreme cytoplasmic [Ca.sup.2+] concentration changes that underlie contraction, remains unknown. Cardiomyocyte plasma membrane [Ca.sup.2+]-ATPase (PMCA) extrudes [Ca.sup.2+] but has little effect on excitation-contraction coupling, suggesting its potential role in controlling [Ca.sup.2+]-dependent signaling effectors such as calcineurin. We generated cardiac-specific inducible PMCA4b transgenic mice that displayed normal global [Ca.sup.2+] transient and cellular contraction levels and reduced cardiac hypertrophy following transverse aortic constriction (TAC) or phenylephrine/Ang II infusion, but showed no reduction in exercise-induced hypertrophy. Transgenic mice were protected from decompensation and fibrosis following long-term TAC. The PMCA4b transgene reduced the hypertrophic augmentation associated with transient receptor potential canonical 3 channel overexpression, but not that associated with activated calcineurin. Furthermore, Pmca4 gene--targeted mice showed increased cardiac hypertrophy and heart failure events after TAC. Physical associations between PMCA4b and calcineurin were enhanced by TAC and by agonist stimulation of cultured neonatal cardiomyocytes. PMCA4b reduced calcineurin nuclear factor of activated T cell--luciferase activity after TAC and in cultured neonatal cardiomyocytes after agonist stimulation. PMCA4b overexpression inhibited cultured cardiomyocyte hypertrophy following agonist stimulation, but much less so in a [Ca.sup.2+] pumping--deficient PMCA4b mutant. Thus, Pmca4b likely reduces the local [Ca.sup.2+] signals involved in reactive cardiomyocyte hypertrophy via calcineurin regulation., Introduction Cardiac hypertrophy is typically characterized by an enlargement of the heart associated with an increase in cardiomyocyte cell volume. Hypertrophy occurs during postnatal development, in response to physiologic stimuli [...]

Muscular dystrophies comprise a diverse group of genetic disorders that lead to muscle wasting and, in many instances, premature death (1). Many mutations that cause muscular dystrophy compromise the support network that connects myofilament proteins within the cell to the basal lamina outside the cell, rendering the sarcolemma more permeable or leaky. Here we show that deletion of the gene encoding cyclophilin D (Ppif) rendered mitochondria largely insensitive to the calcium overload-induced swelling associated with a defective sarcolemma, thus reducing myofiber necrosis in two distinct models of muscular dystrophy. Mice lacking [delta]-sarcoglycan ([Scgd.sup.-/-] mice) showed markedly less dystrophic disease in both skeletal muscle and heart in the absence of Ppif. Moreover, the premature lethality associated with deletion of Lama2, encoding the [alpha]-2 chain of laminin-2, was rescued, as were other indices of dystrophic disease. Treatment with the cyclophilin inhibitor Debio-025 similarly reduced mitochondrial swelling and necrotic disease manifestations in mdx mice, a model of Duchenne muscular dystrophy, and in [Scgd.sup.-/-] mice. Thus, mitochondria'-dependent necrosis represents a prominent disease mechanism in muscular dystrophy, suggesting that inhibition of cyclophilin D could provide a new pharmacologic treatment strategy for these diseases., The muscular dystrophies are inherited disorders that mostly affect striated muscle tissue, resulting in progressive muscle weakness, wasting and, in many instances, premature death (1). Many characterized mutations in humans [...]

Mice null for the gene encoding protein kinase C[alpha] (Prkca), or mice treated with pharmacologic inhibitors of the PKC[alpha]/[beta]/[gamma]/ isoforms, show an augmentation in cardiac contractility that appears to be cardioprotective. However, it remains uncertain if PKC[alpha] itself functions in a myocyte autonomous manner to affect cardioprotection in vivo. Here we generated cardiac myocyte-specific transgenic mice using a tetracycline-inducible system to permit controlled expression of dominant negative PKC[alpha] in the heart. Consistent with the proposed function of PKC[alpha], induction of dominant negative PKC[alpha] expression in the adult heart enhanced baseline cardiac contractility. This increase in cardiac contractility was associated with a partial protection from long-term decompensation and secondary dilated cardiomyopathy after myocardial infarction injury. Similarly, Prkca null mice were also partially protected from infarction-induced heart failure, although the area of infarction injury was identical to controls. Thus, myocyte autonomous inhibition of PKC[alpha] protects the adult heart from decompensation and dilated cardiomyopathy after infarction injury in association with a primary enhancement in contractility. protein kinase C; signaling; myocardial infarction

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 [...]

Thrombospondins are stress-inducible secreted glycoproteins with critical functions in tissue injury and healing. Thrombospondin-4 (Thbs4) is protective in cardiac and skeletal muscle, where it activates an adaptive endoplasmic reticulum (ER) stress response, induces expansion of the ER, and enhances sarcolemmal stability. However, it is unclear if Thbs4 has these protective functions from within the cell, from the extracellular matrix, or from the secretion process itself. In this study, we generated transgenic mice with cardiac cell-specific overexpression of a secretion-defective mutant of Thbs4 to evaluate its exclusive intracellular and secretion-dependent functions. Like wild-type Thbs4, the secretion-defective mutant upregulates the adaptive ER stress response and expands the ER and intracellular vesicles in cardiomyocytes. However, only the secretiondefective Thbs4 mutant produces cardiomyopathy with sarcolemmal weakness and rupture that is associated with reduced adhesion-forming glycoproteins in the membrane. Similarly, deletion of Thbs4 in the mdx mouse model of Duchenne muscular dystrophy enhances cardiomyocyte membrane instability and cardiomyopathy. Finally, overexpression of the secretion-defective Thbs4 mutant in Drosophila, but not wild-type Thbs4, impaired muscle function and sarcomere alignment. These results suggest that transit through the secretory pathway is required for Thbs4 to augment sarcolemmal stability, while ER stress induction and vesicular expansion mediated by Thbs4 are exclusively intracellular processes. [ABSTRACT FROM AUTHOR]

Extracellular matrix production and accumulation stabilize the heart under normal conditions as well as form a protective scar after myocardial infarction injury, although excessive extracellular matrix accumulation with long-standing heart disease is pathological. In the current study we investigate the role of the matricellular protein, transforming growth factor beta-induced (TGFBI), which is induced in various forms of heart disease. Additionally, we sought to understand whether TGFBI is functionally redundant to its closely related family member periostin, which is also induced in the diseased heart. Surgical models of myocardial infarction and cardiac pressure overload were used in mice with genetic loss of Postn and/or Tgfbi to examine the roles of these genes during the fibrotic response. Additionally, cardiac-specific TGFBI transgenic mice were generated and analyzed. We observed that deletion of Tgfbi did not alter cardiac disease after myocardial infarction in contrast to greater ventricular wall rupture in Postn gene-deleted mice. Moreover, Tgfbi and Postn double gene-deleted mice showed a similar post-myocardial infarction disease phenotype as Postn-deleted mice. Over-expression of TGFBI in the hearts of mice had a similar effect as previously shown in mice with periostin over-expression. Thus, TGFBI and periostin act similarly in the heart in affecting fibrosis and disease responsiveness, although TGFBI is not seemingly necessary in the heart after myocardial infarction injury and is fully compensated by the more prominently expressed effector periostin. [ABSTRACT FROM AUTHOR]

Aims L-type Ca2+ channels (LTCCs) in adult cardiomyocytes are localized to t-tubules where they initiate excitation-contraction coupling. Our recent work has shown that a subpopulation of LTCCs found at the surface sarcolemma in caveolae of adult feline cardiomyocytes can also generate a Ca2+ microdomain that activates nuclear factor of activated T-cells signaling and cardiac hypertrophy, although the relevance of this paradigm to hypertrophy regulation in vivo has not been examined. Methods and results Here we generated heart-specific transgenic mice with a putative caveolae-targeted LTCC activator protein that was ineffective in initiating or enhancing cardiac hypertrophy in vivo. We also generated transgenic mice with cardiac-specific overexpression of a putative caveolae-targeted inhibitor of LTCCs, and while this protein inhibited caveolae-localized LTCCs without effects on global Ca2+ handling, it similarly had no effect on cardiac hypertrophy in vivo. Cardiac hypertrophy was elicited by pressure overload for 2 or 12 weeks or with neurohumoral agonist infusion. Caveolae-specific LTCC activator or inhibitor transgenic mice showed no greater change in nuclear factor of activated T-cells activity after 2 weeks of pressure overload stimulation compared with control mice. Conclusion Our results indicate that LTCCs in the caveolae microdomain do not affect cardiac function and are not necessary for the regulation of hypertrophic signaling in the adult mouse heart. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]