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Molkentin, J. D.

Publications and source records attributed to Molkentin, J. D..

14 recordsLinked to original sources

Small GTPases RhoA and RhoC coordinately regulate cardiomyocyte size in the mouse heart

Rho family GTPases regulate cardiomyocyte growth and cytoskeletal organization, yet the overlapping and individual function of the two primary isoforms RhoA and RhoC, remain largely unknown in the heart. Here, we examined cardiac structure and function at baseline and in response to mechanical and neurohumoral stress in mice with cardiomyocyte-specific deletion of Rhoa, global loss of Rhoc, or combined deficiency of both isoforms. Deletion of either Rhoa or Rhoc increased myocardial expression of the other isoform, showing reciprocal compensation. At baseline, Rhoc deficiency produced elongated, narrower cardiomyocytes, whereas additional loss of Rhoa restored cell width but length remained longer. Combined Rhoa/Rhoc deficiency also disrupted intercalated disc protein expression and organization and caused age-dependent cardiac hypertrophy with mild pulmonary congestion despite preserved systolic function. Following pressure overload, loss of either isoform increased hypertrophy and fibrosis compared with control groups, while combining Rhoa/Rhoc deficiency significantly worsened cardiac remodeling and systolic dysfunction. Neurohumoral stimulation with angiotensin II and phenylephrine similarly revealed cooperative control of hypertrophic growth in Rhoa/Rhoc double deleted cardiomyocytes versus either single deletion. These findings identify RhoA and RhoC as partially compensatory effectors of cardiomyocyte growth and ventricular remodeling with stress stimulation, although their individual contributions to hypertrophic growth, structural integrity, and cardiac function depend on both the nature and duration of cardiac stress.

cell biology↗

Deletion of cytoplasmic β/γ-actin in the mouse heart protects from disease by augmenting sarcolemma stability

The actin cytoskeleton of the cardiomyocyte is organized into two structurally and functionally distinct filament networks. The sarcomeric thin filaments, built primarily from cardiac -actin (CA), generate contractile force, whereas a separate subsarcolemmal cytoplasmic network, built from {beta}-actin (Actb gene) and {gamma}-actin (Actg1 gene), lies beneath the sarcolemma and is comparatively understudied in the mature heart. We hypothesized that this cytoplasmic actin network is required for sarcolemmal membrane integrity, signal transduction, and mechanosensing in the adult cardiomyocyte, and we generated cardiomyocyte-specific Actb and Actg1 double-gene deleted mice, using loxP (fl)-targeted Actbfl/fl and Actg1fl/fl alleles combined with an MHC (Myh6) promoter-driven Cre-recombinase transgene, to test this directly. Deletion of cytoplasmic {beta}-actin and {gamma}-actin from cardiomyocytes (Actb/g1fl/fl-Myh6-Cre mice) drove compensatory upregulation of a {gamma}-interferon like stress response with compensatory upregulation of skeletal -actin (SKA) and smooth muscle -actin (SMA) protein in adult cardiomyocytes, without altering baseline cardiac structure or function. To test if lost {beta}-actin and {gamma}-actin in the mouse heart impacts sarcolemmal stability, we crossed Actb/g1fl/fl-Myh6-Cremice onto the dystrophin-deficient mdx background, which is characterized by a fragile sarcolemma. Unexpectedly, hearts from Actb/g1fl/fl-Myh6- Cre; mdx mice showed greater membrane stability than mdx hearts alone with intact Actb/Actg1. We also observed that Actb/g1fl/fl-Myh6-Cremice subjected to chronic pressure overload by transverse aortic constriction (TAC) were protected and developed less cardiac hypertrophy, had better preserved systolic function, and improved survival. Together, these data indicate induction of the cytoplasmic {beta}-actin/{gamma}-actin network in the heart during disease stimulation is maladaptive and weakens the sarcolemma, and 2 downstream mechanisms are considered that could mediate this effect.

cell biology↗

AI-based Predictive Signaling Pathway Profiling in Cardiac Fibrosis Suggests a Novel Combinatorial Treatment Strategy

BackgroundCardiovascular disease (CVD) remains the leading cause of global mortality, with myocardial fibrosis characterized by excessive extracellular matrix (ECM) deposition representing a common endpoint associated with progressive cardiac dysfunction. While studies in animal models of heart disease suggest that preventing or reducing fibrosis can antagonize negative ventricular remodeling, current therapeutic strategies remain clinically limited and ineffective, in part due to an incomplete understanding of the multifactorial nature of the fibrotic process. Methods and ResultsWe employed artificial intelligence (AI) trained from 6,528 cardiac fibrosis publications over the past decade, which suggested 10 nodal signaling pathways underlying the fibrotic process. Single-cell RNA sequencing was used to quantify pathway activities across mouse models and clinical samples encompassing acute and chronic cardiac injury. Dynamic enrichment analysis revealed 2 critical pathways involved in acute myocardial infarction (MI) injury driven temporally-regulated fibrosis whereby Janus kinase - Signal transducer and activator of transcription (JAK-STAT) signaling peaked early (day 7) while transforming growth factor-{beta} (TGF-{beta}) pathway activity peaked mid-phase (day 14). With more chronically-driven fibrosis, JAK-STAT signaling again emerged, which this time was persistently active during chronic transverse aortic constriction (TAC) injury. Experimentally, single-cell analysis identified a pathogenic myofibroblast subpopulation characterized by high JAK-STAT signaling and ECM secretion capacity. Fibroblast-specific Jak1/2 gene-deleted mice significantly reduced TAC-induced fibrosis by blocking myofibroblast formation from this subpopulation, although these same mice failed to show attenuated fibrosis following MI injury, suggesting a pathway that would be ideal to therapeutically target for chronic fibrosis without affecting necessary acute scar formation. Indeed, based on the predictive AI driven algorithm, a temporal inhibitory strategy was generated for JAK-STAT and TGF-{beta} that permitted compensatory scar formation while more effectively preventing progressive fibrosis and worsened cardiac function versus either singular pathway or chronic antagonism. ConclusionHere we employed the wealth of past signaling pathway data underlying cardiac fibrosis to train an AI model, which suggested an optimized approach of inhibiting 2 nodal signaling pathways but with differential timing as a more effective therapeutic strategy that reduces pathologic cardiac fibrosis without negatively impacting compensatory fibrotic activity with acute MI injury. The therapeutic approach involves altering the timing of JAK-STAT signaling blockade with ruxolitinib (RUX) in combination with delayed and temporary TGF-{beta} signaling inhibition post MI injury with pirfenidone (PFD).

cell biology↗

Cardiomyocyte-expressed TGFβ signals to fibroblasts to program early heart maturation and adult myocyte identity

Transforming growth factor {beta} (TGF{beta}) is a secreted growth factor that is sequestered to the extracellular matrix (ECM) as a latent complex. In adult disease TGF{beta} release in the heart transforms fibroblasts into a differentiated state that synthesizes more ECM. However, it is not known how TGF{beta} functions in the early developing heart to impact resident fibroblasts. Here, we observe that deletion of the Tgfb1, Tgfb2, and Tgfb3 genes (TGF{beta} ligands) from cardiomyocytes in the early developing heart results in cardiac dysfunction by 6 weeks of age with altered fibroblast activity and altered ECM content. Early postnatal hearts from Tgfb1/2/3 cardiomyocyte-deleted mice are dysmorphic and cardiac fibroblasts have incorrect activity and produce inappropriate ECM with reduced stiffness. Gene expression profiling of hearts from myocyte-specific Tgfb1/2/3 deleted mice reveal defects in both cardiomyocyte and fibroblast maturation with ectopic expression of multiple skeletal muscle-specific genes beginning at embryonic day 17.5 and progressing with age. However, cardiomyocyte-specific deletion of TGF{beta} receptors I/II encoding genes (Tgfbr1/2) or Smad2/3 encoding genes (Smad2/3) do not recapitulate this phenotype suggesting that TGF{beta} directly programs early heart fibroblast development that in turn specifies cardiomyocyte maturation. Importantly, Col1a2-/-;Col6a2-/- mice with defective cardiac ECM stiffness, mice lacking cardiomyocyte Itgb1 with reduced ECM load sensing, and Tcf21-/- embryos at E17.5 lacking cardiac fibroblasts each fail to generate the same pathologic ECM program with ectopic cardiomyocyte differentiation observed with Tgfb1/2/3 myocyte-specific deletion. These and additional results indicate that TGF{beta} generated by cardiomyocytes in the embryonic heart mediates fibroblast differentiation that co-evolves the ECM environment that in turn programs cardiomyocyte maturation to establish their identity.

developmental biology↗

Atad3 is Essential for Mitochondrial Permeability Transition Pore Opening and Cardiac Ischemia Reperfusion Injury.

The molecular identity of the mitochondrial permeability transition pore (mPTP) has remained elusive since the discovery of its existence over 75 years ago. Despite the numerous candidate proteins proposed, none have withstood genetic ablation, leaving them relegated to auxiliary regulatory roles. To date, no essential mPTP component has been identified. Here, we establish ATAD3 as the first essential component of the mPTP. Genetic deletion of Atad3 in cardiomyocytes and hepatocytes renders heart and liver mitochondria incapable of undergoing Ca2+-induced mPTP-dependent swelling. Moreover, these mitochondria exhibit the highest Ca2+ retention capacity ever reported following genetic perturbation of the mPTP. Furthermore, patch-clamp recordings of recombinant ATAD3a in liposomes reveal intrinsic channel activity. Given the established role of mPTP-dependent necrosis in driving ischemia/reperfusion (I/R) injury, we show that cardiac-specific Atad3 deletion markedly reduces infarct size following I/R, with no additive protection from cyclosporine A. Together, these findings establish ATAD3 as an core, putative pore-forming component essential for mPTP opening and mPTP-dependent necrosis, resolving a long-standing mystery in mitochondrial biology.

cell biology↗

Myocardial Endoglin Regulates Cardiomyocyte Proliferation and Cardiac Regeneration

The mammalian heart loses almost all its regenerative potential in the first week of life due to the cessation of the ability of cardiomyocytes to proliferate. In recent years, a number of regulators of cardiomyocyte proliferation have been identified. Despite this, a clear understanding of the regulatory pathways that control cardiomyocyte proliferation and cardiac regeneration is lacking, and there are likely additional regulators to be discovered. Here, we performed a genome-wide screen on fetal murine cardiomyocytes to identify potential novel regulators of cardiomyocyte proliferation. Endoglin was identified as an inhibitor of cardiomyocyte proliferation in vitro. Endoglin knock-down resulted in enhanced DNA synthesis, cardiomyocyte mitosis and cytokinesis in mouse, rat and human cardiomyocytes. Using gene-targeted mice, we confirmed myocardial Endoglin to be important in cardiomyocyte proliferation and cardiac regeneration using gene-targeted mice. Mechanistically, we show that Smad signaling is required for the endoglin-mediated anti-proliferative effects. Our results identify the TGF-{beta} coreceptor Endoglin as a regulator of cardiac regeneration and cardiomyocyte proliferation. SummaryHigh-content function screening is used to identify a novel inhibitor of cardiomyocyte proliferation which can promote mammalian cardiac regeneration.

genetics↗

Cardiac fibroblasts regulate cardiomyocyte hypertrophy through dynamic regulation of type I collagen

RationaleCardiomyocytes and fibroblasts in the heart communicate through both secreted growth factors as well as through sensing the structural properties of the extracellular matrix that each helps generate. Previous studies have shown that defects in fibroblast activity during disease stimulation result in altered cardiomyocyte hypertrophy, although the role that collagen might play in this communication is unknown. ObjectiveHere we investigated how type I collagen maturation and disease-responsive matrix expansion in the heart by cardiac fibroblasts impacts cardiac fibrosis and cardiomyocyte hypertrophy. Methods and ResultsWe generated and characterized Col1a2-/- mice using standard gene-targeting. Col1a2-/- mice were viable, although by young adulthood their hearts showed alterations in extracellular matrix mechanical properties, as well as an unanticipated activation of cardiac fibroblasts and induction of a progressive fibrotic response. This included increases in fibroblast number and a progressive cardiac hypertrophy, with reduced functional performance by 9 months. Col1a2-loxP targeted mice were also generated and crossed with the tamoxifen-inducible Postn-MerCreMer knock-in mice to delete the Col1a2 gene in myofibroblasts post-pressure overload injury, to more specifically implicate fibroblasts as effectors of cardiomyocyte hypertrophy in vivo. Opposite to the gradual induction of cardiac hypertrophy observed in germline Col1a2-/- mice as they matured developmentally, adult fibroblast-specific deletion of Col1a2 during pressure overload protected these mice from cardiac hypertrophy in the first week with a delayed fibrotic response. However, this reduction in hypertrophy due to myofibroblast-specific Col1a2 deletion was gradually lost over 2 and 6 weeks of pressure overload as augmented fibrosis returned. ConclusionsDefective type I collagen in the developing heart alters the structural integrity of the extracellular matrix that leads to fibroblast expansion, activation, fibrosis and hypertrophy with progressive cardiomyopathy in adulthood. However, acute deletion of type I collagen production for the first time in the adult heart during pressure overload prevents ECM expansion and inhibits cardiomyocyte hypertrophy, while gradual restoration of fibrosis again permitted hypertrophy comparable to controls.

cell biology↗

Loss of Acta2 in cardiac fibroblasts does not affect myofibroblast differentiation or cardiac repair after myocardial infarction

In response to myocardial infarction (MI), quiescent cardiac fibroblasts differentiate into myofibroblasts mediating tissue repair in the infarcted area. One of the most widely accepted markers of myofibroblast differentiation is the expression of Acta2 which encodes smooth muscle alpha-actin (SMA) that is assembled into stress fibers. However, the requirement of Acta2 in the myofibroblast differentiation of cardiac fibroblasts and its role in post-MI cardiac repair were still not known. To answer these questions, we generated a tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse line. Surprisingly, mice that lacked Acta2 in cardiac fibroblasts had a normal survival rate after MI. Moreover, Acta2 deletion did not affect the function or overall histology of infarcted hearts. No difference was detected in the proliferation, migration, or contractility between WT cardiac fibroblasts and Acta2-null cardiac myofibroblasts. Additional analysis identified that Acta2-null cardiac myofibroblasts had a normal total filamentous actin level and total actin level. Acta2 deletion caused a unique compensatory increase in the transcription level of Actg2 and a possible increase in the protein abundance of cytoplasmic actin isoforms. In conclusion, the deletion of Acta2 does not prevent the myofibroblast differentiation of cardiac fibroblasts or affect the post-MI cardiac repair, and the increase in the expression of non-SMA actin isoforms and the functional redundancy between actin isoforms are likely able to compensate for the loss of Acta2 in cardiac myofibroblasts.

molecular biology↗

S-Palmitoylation-Dependent Regulation of Cardiomyocyte Rac1 Signaling Activity and Cardiac Hypertrophy

S-palmitoylation is a reversible lipid modification that regulates trafficking, localization, activity, and/or stability of protein substrates by serving as a fatty acid anchor to cell membranes. However, S-palmitoylation-dependent control of signal transduction in cardiomyocytes and its effects on cardiac physiology are not well understood. We performed an in vivo gain-of-function screen of zinc finger Asp-His-His-Cys (zDHHC) family S-acyl transferases that catalyze S-palmitoylation and identified the Golgi-localized enzyme zDHHC3 as a critical regulator of cardiac maladaptation. The closely-related enzyme, zDHHC7, also induced severe cardiomyopathy but this effect was not observed with overexpression of plasma membrane enzyme zDHHC5, endoplasmic reticulum enzyme zDHHC6, or Golgi enzyme zDHHC13. To identify effectors that may underlie zDHHC3-induced cardiomyopathy we performed quantitative site-specific S-acyl proteomics in zDHHC3-overexpressing cells that revealed the small GTPase Rac1 as a novel substrate. We generated cardiomyocyte-specific transgenic mice overexpressing zDHHC3, which develop severe cardiac disease. Cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice are preceded by enhanced S-palmitoylation of Rac1 and induction of additional Rho family small GTPases including RhoA, Cdc42, and the Rho family-specific chaperone RhoGDI. In contrast, transgenic mice overexpressing an enzymatically-dead mutant of zDHHC3 do not exhibit this profound induction of RhoGTPase signaling or develop cardiac disease. Rac1 S-palmitoylation, plasma membrane localization, activity, and downstream hypertrophic signaling were substantially increased in zDHHC3 overexpressing hearts. Taken together, these data suggest inhibition of zDHHC3/7 S-acyl transferase activity at the cardiomyocyte Golgi or disruption of Rac1 S-palmitoylation as novel therapeutic strategies to treat cardiac disease or other diseases associated with enhanced RhoGTPase signaling.

molecular biology↗

Satellite cell depletion in early adulthood attenuates muscular dystrophy pathogenesis

Satellite cells are skeletal muscle resident stem cells that regenerate adult myofibers following an acute injury to muscle. Despite the assumption that the loss of satellite cells would be detrimental in a chronic regeneration-inducing muscle disease such as muscular dystrophy, this assumption has never been tested using mouse genetics. Here we generated a novel model of satellite cell ablation and crossed it with mouse models of muscular dystrophy to directly investigate how critical these cells are in maintaining muscle during a chronic degenerative disorder. Satellite cell deletion in 2-week-old young dystrophic mice provided noticeable improvements in histopathology and function, although at this early timepoint it was utimately detrimental because muscle size was not sufficient to permit survival. However, depletion of satellite cells beginning at 2 months of age in dystrophic mice provided similar histological and functional improvements but without compromising muscle size. The improved profile showed fewer damaged fibers, less myofiber central nucleation, increased sarcolemma integrity, decreased fibrosis and a dramatic size increase in the remaining myofibers. At the functional level, young adult dystrophic mice lacking satellite cells performed significantly better than those with satellite cells when exercised on a treadmill. Thus, loss of satellite cells during early adulthood in dystrophic mice produces an unexpected protective effect.

molecular biology↗

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

The mitochondrial permeability transition pore (MPTP) has resisted molecular identification for decades. The original model of the MPTP had the adenine nucleotide translocator (ANT) as the inner membrane pore-forming component. Indeed, reconstitution experiments showed that recombinant or purified ANT generates MPTP-like pores in lipid bilayers. This model was challenged when mitochondria from Ant1/2 double null mouse liver still showed MPTP activity. Because mice contain and express 3 Ant genes, here we reinvestigated the genetic basis for the ANTs as comprising the MPTP. Liver mitochondria from Ant1, Ant2, and Ant4 deficient mice were highly refractory to Ca2+-induced MPT, and when also given cyclosporine A, MPT 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 MPT. Finally, inner membrane patch clamping in mitochondria from Ant1, Ant2 and Ant4 triple null mouse embryonic fibroblasts (MEFs) showed a loss of MPT-like pores. Our findings suggest a new model of MPT consisting of two distinct molecular components, one of which is the ANTs and the other of which is unknown but requires CypD.\n\nOne Sentence SummaryGenetic deletion of Ant1/2/4 and Ppif in mice fully inhibits the mitochondrial permeability transition pore

cell biology↗

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

Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day1-3 despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biologic effect4-6. 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 ischemic injury7-9. Here we examined the mechanistic basis for cell therapy in mice after ischemia/reperfusion (I/R) injury, and while heart function was enhanced, it was not associated with new cardiomyocyte production. Cell therapy improved heart function through an acute sterile immune response characterized by the temporal and regional induction of CCR2+ and CX3CR1+ macrophages. Here we observed that intra-cardiac injection of 2 distinct types of progenitor cells, freeze/thaw-killed cells or a chemical inducer of the innate immune response similarly induced regional CCR2+ and CX3CR1+ macrophage accumulation and provided functional rejuvenation to the I/R-injured heart. Mechanistically, this selective macrophage response altered cardiac fibroblast activity and reduced border zone extracellular matrix (ECM) content 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 mechanical properties of the infarcted area of the heart. Such results suggest a re-evaluation of strategies underlying cardiac cell therapy in current and planned human clinical trials.

cell biology↗

Evidence for minimal cardiogenic potential of Sca-1 positive cells in the adult mouse heart

BackgroundDespite modern pharmacotherapy, heart failure remains a major medical burden. The heart has a limited regenerative capacity, and bolstering regeneration might represent new therapeutic approaches for heart failure patients. Various progenitor cells in the heart have been proposed to have cardiomyogenic properties, but this evidence is based mostly on cell culture and transplantation studies. One population of interest is characterized by the expression of Stem Cell Antigen-1 (Sca-1). Here we tested the hypothesis that Sca-1+ cells are endogenous progenitors for cardiomyocytes in the adult heart.\n\nMethodsWe evaluated the innate cardiogenic potential of Sca-1+ cells in vivo by generating a novel mouse model to genetically lineage-trace the fate of Sca-1 expressing cells. This was accomplished by introducing a tamoxifen-inducible Cre-recombinase into the Sca-1 locus (Sca-1mCm/+). Crossing this mouse line to a Cre-dependent tdTomato reporter line allowed for genetic lineage-tracing of endogenous Sca-1+ cells (Sca-1mCmR26tdTomato). The frequency of Sca-1+ cardiomyocytes was quantified from dispersed cell preparations and confirmed by in situ histology.\n\nResultsWe validated the genetic lineage tracing mouse model in bone marrow and heart. Unlike previous publications suggesting significant cardiogenic potential, we found that less than 0.02% of cardiomyocytes per year were derived from Sca-1+ cells in the adult heart under homeostatic conditions. At six months after myocardial infarction, we found less than 0.01% of cardiomyocytes were derived from Sca-1+ cells.\n\nConclusionOur results show that Sca-1+ cells in the adult heart have minimal cardiogenic potential under homeostatic conditions or in response to myocardial infarction.

cell biology↗

Mitogen-Activated Protein Kinase-Dependent Fiber-Type Regulation in Skeletal Muscle

Mitogen-activated protein kinases (MAPK) are conserved protein kinases that regulate a diverse array of cellular activities. Stress or mitogenic signals activate three primary branches of the greater MAPK cascade, each of which consists of a phosphorylation-dependent array of successively acting kinases. The extracellular signal-regulated kinase 1/2 (ERK1/2) branch is regulated by growth factory signaling at the cell membrane, leading to phosphorylation of the dual-specificity kinase MEK1, which is dedicated to ERK1/2 phosphorylation. Previous studies have established a link between MAPK activation and endurance exercise, but whether a single MAPK is responsible for establishing muscle metabolic fate is unclear. Using mouse genetics we observed that muscle-specific expression of a constitutively active MEK1 promotes greater ERK1/2 signaling that mediates fiber-type switching in mouse skeletal muscle to a slow, oxidative phenotype with type I myosin heavy chain expression. Induced expression of the activated MEK1 mutant using either a MyoD-Cre or myosin light chain-Cre strategy equally increased the number of type I fibers in skeletal muscle with significantly reduced size compared to controls. Moreover, activation of MEK1 in mature myofibers of an adult mouse using a transgene containing a tamoxifen inducible MerCreMer cDNA under the control of a skeletal -actin promoter produced a similar phenotype of switching towards a slow-oxidative program. Physiologic assessment of mice with greater skeletal muscle slow-oxidative fibers showed enhanced metabolic activity and oxygen consumption with greater fatigue resistance of individual muscles. In summary, these results show that sustained MEK1-ERK1/2 activity in skeletal muscle produces a fast-to-slow fiber-type switch, suggesting that modulation of this signaling pathway may represent a therapeutic approach to enhance the long-term metabolic effectiveness of muscle in vivo.

developmental biology↗