MEK1-ERK1/2 signaling regulates the cardiomyocyte non-sarcomeric actin cytoskeletal network.

During select pathological conditions, the heart can hypertrophy and remodel in either a dilated or concentric ventricular geometry, which is associated with lengthening or widening of cardiomyocytes, respectively. The mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinase 1 and 2 (ERK1/2) pathway has been implicated in these differential types of growth such that cardiac overexpression of activated MEK1 causes profound concentric hypertrophy and cardiomyocyte thickening, while genetic ablation of the genes encoding ERK1/2 in the mouse heart causes dilation and cardiomyocyte lengthening. However, the mechanisms by which this kinase signaling pathway controls cardiomyocyte directional growth as well as its downstream effectors are poorly understood. To investigate this, we conducted an unbiased phosphoproteomic screen in cultured neonatal rat ventricular myocytes treated with an activated MEK1 adenovirus, the MEK1 inhibitor U0126, or an eGFP adenovirus control. Bioinformatic analysis identified cytoskeletal-related proteins as the largest subset of differentially phosphorylated proteins. Phos-tag and traditional Western blotting were performed to confirm that many cytoskeletal proteins displayed changes in phosphorylation with manipulations in MEK1-ERK1/2 signaling. From this, we hypothesized that the actin cytoskeleton would be changed in vivo in the mouse heart. Indeed, we found that activated MEK1 transgenic mice and gene-deleted mice lacking ERK1/2 protein had enhanced non-sarcomeric actin expression in cardiomyocytes compared with wild-type control hearts. Consistent with these results, cytoplasmic ß- and γ-actin were increased at the subcortical intracellular regions of adult cardiomyocytes. Together, these data suggest that MEK1-ERK1/2 signaling influences the non-sarcomeric cytoskeletal actin network, which may be important for facilitating the growth of cardiomyocytes in length and/or width.NEW & NOTEWORTHY Here, we performed an unbiased analysis of the total phosphoproteome downstream of MEK1-ERK1/2 kinase signaling in cardiomyocytes. Pathway analysis suggested that proteins of the non-sarcomeric cytoskeleton were the most differentially affected. We showed that cytoplasmic ß-actin and γ-actin isoforms, regulated by MEK1-ERK1/2, are localized to the subcortical space at both lateral membranes and intercalated discs of adult cardiomyocytes suggesting how MEK1-ERK1/2 signaling might underlie directional growth of adult cardiomyocytes.

MCUb is an inducible regulator of calcium-dependent mitochondrial metabolism and substrate utilization in muscle.

Mitochondria use the electron transport chain to generate high-energy phosphate from oxidative phosphorylation, a process also regulated by the mitochondrial Ca2+ uniporter (MCU) and Ca2+ levels. Here, we show that MCUb, an inhibitor of MCU-mediated Ca2+ influx, is induced by caloric restriction, where it increases mitochondrial fatty acid utilization. To mimic the fasted state with reduced mitochondrial Ca2+ influx, we generated genetically altered mice with skeletal muscle-specific MCUb expression that showed greater fatty acid usage, less fat accumulation, and lower body weight. In contrast, mice lacking Mcub in skeletal muscle showed increased pyruvate dehydrogenase activity, increased muscle malonyl coenzyme A (CoA), reduced fatty acid utilization, glucose intolerance, and increased adiposity. Mechanistically, pyruvate dehydrogenase kinase 4 (PDK4) overexpression in muscle of Mcub-deleted mice abolished altered substrate preference. Thus, MCUb is an inducible control point in regulating skeletal muscle mitochondrial Ca2+ levels and substrate utilization that impacts total metabolic balance.

A human FLII gene variant alters sarcomeric actin thin filament length and predisposes to cardiomyopathy.

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 predisposes 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.

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.

Thbs1 induces lethal cardiac atrophy through PERK-ATF4 regulated autophagy.

The thrombospondin (Thbs) family of secreted matricellular proteins are stress- and injury-induced mediators of cellular attachment dynamics and extracellular matrix protein production. Here we show that Thbs1, but not Thbs2, Thbs3 or Thbs4, induces lethal cardiac atrophy when overexpressed. Mechanistically, Thbs1 binds and activates the endoplasmic reticulum stress effector PERK, inducing its downstream transcription factor ATF4 and causing lethal autophagy-mediated cardiac atrophy. Antithetically, Thbs1-/- mice develop greater cardiac hypertrophy with pressure overload stimulation and show reduced fasting-induced atrophy. Deletion of Thbs1 effectors/receptors, including ATF6α, CD36 or CD47 does not diminish Thbs1-dependent cardiac atrophy. However, deletion of the gene encoding PERK in Thbs1 transgenic mice blunts the induction of ATF4 and autophagy, and largely corrects the lethal cardiac atrophy. Finally, overexpression of PERK or ATF4 using AAV9 gene-transfer similarly promotes cardiac atrophy and lethality. Hence, we identified Thbs1-mediated PERK-eIF2α-ATF4-induced autophagy as a critical regulator of cardiomyocyte size in the stressed heart.

MCUb Induction Protects the Heart From Postischemic Remodeling.

RATIONALE: Mitochondrial Ca2+ loading augments oxidative metabolism to match functional demands during times of increased work or injury. However, mitochondrial Ca2+ overload also directly causes mitochondrial rupture and cardiomyocyte death during ischemia-reperfusion injury by inducing mitochondrial permeability transition pore opening. The MCU (mitochondrial Ca2+ uniporter) mediates mitochondrial Ca2+ influx, and its activity is modulated by partner proteins in its molecular complex, including the MCUb subunit. OBJECTIVE: Here, we sought to examine the function of the MCUb subunit of the MCU-complex in regulating mitochondria Ca2+ influx dynamics, acute cardiac injury, and long-term adaptation after ischemic injury. METHODS AND RESULTS: Cardiomyocyte-specific MCUb overexpressing transgenic mice and Mcub gene-deleted (Mcub-/-) mice were generated to dissect the molecular function of this protein in the heart. We observed that MCUb protein is undetectable in the adult mouse heart at baseline, but mRNA and protein are induced after ischemia-reperfusion injury. MCUb overexpressing mice demonstrated inhibited mitochondrial Ca2+ uptake in cardiomyocytes and partial protection from ischemia-reperfusion injury by reducing mitochondrial permeability transition pore opening. Antithetically, deletion of the Mcub gene exacerbated pathological cardiac remodeling and infarct expansion after ischemic injury in association with greater mitochondrial Ca2+ uptake. Furthermore, hindlimb remote ischemic preconditioning induced MCUb expression in the heart, which was associated with decreased mitochondrial Ca2+ uptake, collectively suggesting that induction of MCUb protein in the heart is protective. Similarly, mouse embryonic fibroblasts from Mcub-/- mice were more sensitive to Ca2+ overload. CONCLUSIONS: Our studies suggest that Mcub is a protective cardiac inducible gene that reduces mitochondrial Ca2+ influx and permeability transition pore opening after ischemic injury to reduce ongoing pathological remodeling.

Probing Pedomorphy and Prolonged Lifespan in Naked Mole-Rats and Dwarf Mice.

Pedomorphy, maintenance of juvenile traits throughout life, is most pronounced in extraordinarily long-lived naked mole-rats. Many of these traits (e.g., slow growth rates, low hormone levels, and delayed sexual maturity) are shared with spontaneously mutated, long-lived dwarf mice. Although some youthful traits likely evolved as adaptations to subterranean habitats (e.g., thermolability), the nature of these intrinsic pedomorphic features may also contribute to their prolonged youthfulness, longevity, and healthspan.

ERK1/2 signaling induces skeletal muscle slow fiber-type switching and reduces muscular dystrophy disease severity.

Mitogen-activated protein kinase (MAPK) signaling consists of an array of successively acting kinases. The extracellular signal-regulated kinases 1/2 (ERK1/2) are major components of the greater MAPK cascade that transduce growth factor signaling at the cell membrane. Here we investigated ERK1/2 signaling in skeletal muscle homeostasis and disease. Using mouse genetics, we observed that the muscle-specific expression of a constitutively active MEK1 mutant promotes greater ERK1/2 signaling that mediates fiber-type switching to a slow, oxidative phenotype with type I myosin heavy chain expression. Using a conditional and temporally regulated Cre strategy as well as Mapk1 (ERK2) and Mapk3 (ERK1) genetically targeted mice, MEK1-ERK2 signaling was shown to underlie this fast-to-slow fiber type switching in adult skeletal muscle as well as during development. Physiologic assessment of these activated MEK1-ERK1/2 mice showed enhanced metabolic activity and oxygen consumption with greater muscle fatigue resistance. Moreover, induction of MEK1-ERK1/2 signaling increased dystrophin and utrophin protein expression in a mouse model of limb-girdle muscle dystrophy and protected myofibers from damage. In summary, sustained MEK1-ERK1/2 activity in skeletal muscle produces a fast-to-slow fiber-type switch that protects from muscular dystrophy, suggesting a therapeutic approach to enhance the metabolic effectiveness of muscle and protect from dystrophic disease.

Supporting the heart: Functions of the cardiomyocyte's non-sarcomeric cytoskeleton.

The non-contractile cytoskeleton in cardiomyocytes is comprised of cytoplasmic actin, microtubules, and intermediate filaments. In addition to providing mechanical support to these cells, these structures are important effectors of tension-sensing and signal transduction and also provide networks for the transport of proteins and organelles. The majority of our knowledge on the function and structure of these cytoskeletal networks comes from research on proliferative cell types. However, in recent years, researchers have begun to show that there are important cardiomyocyte-specific functions of the cytoskeleton. Here we will discuss the current state of cytoskeletal biology in cardiomyocytes, as well as research from other cell types, that together suggest there is a wealth of knowledge on cardiac health and disease waiting to be uncovered through exploration of the complex signaling networks of cardiomyocyte non-sarcomeric cytoskeletal proteins.

Overlapping and differential functions of ATF6α versus ATF6ß in the mouse heart.

Hemodynamic stress on the mammalian heart results in compensatory hypertrophy and activation of the unfolded protein response through activating transcription factor 6α (ATF6α) in cardiac myocytes, but the roles of ATF6α or the related transcription factor ATF6ß in regulating this hypertrophic response are not well-understood. Here we examined the effects of loss of ATF6α or ATF6ß on the cardiac response to pressure overload. Mice gene-deleted for Atf6 or Atf6b were subjected to 2 weeks of transverse aortic constriction, and each showed a significant reduction in hypertrophy with reduced expression of endoplasmic reticulum (ER) stress-associated proteins compared with controls. However, with long-term pressure overload both Atf6 and Atf6b null mice showed enhanced decompensation typified by increased heart weight, pulmonary edema and reduced function compared to control mice. Our subsequent studies using cardiac-specific transgenic mice expressing the transcriptionally active N-terminus of ATF6α or ATF6ß revealed that these factors control overlapping gene expression networks that include numerous ER protein chaperones and ER associated degradation components. This work reveals previously unappreciated roles for ATF6α and ATF6ß in regulating the pressure overload induced cardiac hypertrophic response and in controlling the expression of genes that condition the ER during hemodynamic stress.

The naked mole-rat exhibits an unusual cardiac myofilament protein profile providing new insights into heart function of this naturally subterranean rodent.

The long-lived, hypoxic-tolerant naked mole-rat well-maintains cardiac function over its three-decade-long lifespan and exhibits many cardiac features atypical of similar-sized laboratory rodents. For example, they exhibit low heart rates and resting cardiac contractility, yet have a large cardiac reserve. These traits are considered ecophysiological adaptations to their dank subterranean atmosphere of low oxygen and high carbon dioxide levels and may also contribute to negligible declines in cardiac function during aging. We asked if naked mole-rats had a different myofilament protein signature to that of similar-sized mice that commonly show both high heart rates and high basal cardiac contractility. Adult mouse ventricles predominantly expressed α-myosin heavy chain (97.9 ± 0.4%). In contrast, and more in keeping with humans, ß myosin heavy chain was the dominant isoform (79.0 ± 2.0%) in naked mole-rat ventricles. Naked mole-rat ventricles diverged from those of both humans and mice, as they expressed both cardiac and slow skeletal isoforms of troponin I. This myofilament protein profile is more commonly observed in mice in utero and during cardiomyopathies. There were no species differences in phosphorylation of cardiac myosin binding protein-C or troponin I. Phosphorylation of both ventricular myosin light chain 2 and cardiac troponin T in naked mole-rats was approximately half that observed in mice. Myofilament function was also compared between the two species using permeabilized cardiomyocytes. Together, these data suggest a cardiac myofilament protein signature that may contribute to the naked mole-rat's suite of adaptations to its natural subterranean habitat.

TGFBI functions similar to periostin but is uniquely dispensable during cardiac injury.

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.

Reaching Out to Send a Message: Proteins Associated with Neurite Outgrowth and Neurotransmission are Altered with Age in the Long-Lived Naked Mole-Rat.

Aging is the greatest risk factor for developing neurodegenerative diseases, which are associated with diminished neurotransmission as well as neuronal structure and function. However, several traits seemingly evolved to avert or delay age-related deterioration in the brain of the longest-lived rodent, the naked mole-rat (NMR). The NMR remarkably also exhibits negligible senescence, maintaining an extended healthspan for ~75 % of its life span. Using a proteomic approach, statistically significant changes with age in expression and/or phosphorylation levels of proteins associated with neurite outgrowth and neurotransmission were identified in the brain of the NMR and include: cofilin-1; collapsin response mediator protein 2; actin depolymerizing factor; spectrin alpha chain; septin-7; syntaxin-binding protein 1; synapsin-2 isoform IIB; and dynamin 1. We hypothesize that such changes may contribute to the extended lifespan and healthspan of the NMR.

And the beat goes on: maintained cardiovascular function during aging in the longest-lived rodent, the naked mole-rat.

The naked mole-rat (NMR) is the longest-lived rodent known, with a maximum lifespan potential (MLSP) of >31 years. Despite such extreme longevity, these animals display attenuation of many age-associated diseases and functional changes until the last quartile of their MLSP. We questioned if such abilities would extend to cardiovascular function and structure in this species. To test this, we assessed cardiac functional reserve, ventricular morphology, and arterial stiffening in NMRs ranging from 2 to 24 years of age. Dobutamine echocardiography (3 µg/g ip) revealed no age-associated changes in left ventricular (LV) function either at baseline or with exercise-like stress. Baseline and dobutamine-induced LV pressure parameters also did not change. Thus the NMR, unlike other mammals, maintains cardiac reserve with age. NMRs showed no cardiac hypertrophy, evidenced by no increase in cardiomyocyte cross-sectional area or LV dimensions with age. Age-associated arterial stiffening does not occur since there are no changes in aortic blood pressures or pulse-wave velocity. Only LV interstitial collagen deposition increased 2.5-fold from young to old NMRs (P < 0.01). However, its effect on LV diastolic function is likely minor since NMRs experience attenuated age-related increases in diastolic dysfunction in comparison with other species. Overall, these findings conform to the negligible senescence phenotype, as NMRs largely stave off cardiovascular changes for at least 75% of their MLSP. This suggests that using a comparative strategy to find factors that change with age in other mammals but not NMRs could provide novel targets to slow or prevent cardiovascular aging in humans.

Cardiac function of the naked mole-rat: ecophysiological responses to working underground.

The naked mole-rat (NMR) is a strictly subterranean rodent with a low resting metabolic rate. Nevertheless, it can greatly increase its metabolic activity to meet the high energetic demands associated with digging through compacted soils in its xeric natural habitat where food is patchily distributed. We hypothesized that the NMR heart would naturally have low basal function and exhibit a large cardiac reserve, thereby mirroring the species' low basal metabolism and large metabolic scope. Echocardiography showed that young (2-4 yr old) healthy NMRs have low fractional shortening (28 ± 2%), ejection fraction (43 ± 2%), and cardiac output (6.5 ± 0.4 ml/min), indicating low basal cardiac function. Histology revealed large NMR cardiomyocyte cross-sectional area (216 ± 10 µm(2)) and cardiac collagen deposition of 2.2 ± 0.4%. Neither of these histomorphometric traits was considered pathological, since biaxial tensile testing showed no increase in passive ventricular stiffness. NMR cardiomyocyte fibers showed a low degree of rotation, contributing to the observed low NMR cardiac contractility. Interestingly, when the exercise mimetic dobutamine (3 µg/g ip) was administered, NMRs showed pronounced increases in fractional shortening, ejection fraction, cardiac output, and stroke volume, indicating an increased cardiac reserve. The relatively low basal cardiac function and enhanced cardiac reserve of NMRs are likely to be ecophysiological adaptations to life in an energetically taxing environment.

Getting to the heart of the matter: age-related changes in diastolic heart function in the longest-lived rodent, the naked mole rat.

The naked mole rat is an extremely long-lived (>31 years) small (35 g) rodent. Moreover, it maintains good health for most of its long life. We hypothesized that naked mole rats also show attenuated cardiac aging. With age, cardiac muscle can become less compliant, causing a decline in early diastolic filling (E) and a compensatory increase in atrial contraction-induced late filling (A). This results in decreased left ventricular E/A ratio. Doppler imaging showed no significant differences in E/A ratios (p = .48) among old (18-20 years) breeders and nonbreeders despite differences in estrogen levels. A cross-sectional study of 1- to 20-year-old naked mole rats (n = 76) revealed that E/A ratios declined with age in females (n = 40; p = .002) but not in males (n = 36; p = 0.45). Despite this, neither gender shows increased morbidity or mortality with age. These findings suggest that, notwithstanding the previously observed high lipid peroxidation in heart tissue, NMRs must possess mechanisms to stave off progression to fatal cardiac disease.