BACKGROUND:Danon disease is a rare, X-linked, monogenic cardiomyopathy caused by mutations in the lysosomal-associated membrane 2 gene (LAMP2), which encodes the LAMP2 protein. In male patients, the predominant phenotype is progressive cardiac hypertrophy, cardiac dysfunction, and early death. There are no directed therapies for the disease. METHODS:In this phase 1 study, we evaluated the safety and efficacy of a single infusion of RP-A501, a recombinant adeno-associated virus serotype 9 containing the transgene LAMP2B, which encodes an isoform of LAMP2. The primary outcomes were the safety and toxic effects of RP-A501, myocardial LAMP2 transduction and protein expression, stabilization of or reduction in heart-failure symptoms, and stabilization of or improvement in cardiac structure and function. Key secondary outcomes were sustained reduction in or stabilization of symptoms, immunologic response to RP-A501, end-stage heart failure, and overall survival. Exploratory outcomes included improvement in serologic markers of cardiac disease, patient-reported outcomes, and quality-of-life assessments. RESULTS:RP-A501 infusion was administered to seven male patients with Danon disease: five who were 15 years of age or older and two who were between 11 and 14 years of age. All the patients received a transient immunomodulatory regimen of prednisone, tacrolimus or sirolimus, and rituximab. Phase 1 data over 24 to 54 months, including interim data from a long-term follow-up study, are reported here. One patient had complement-mediated thrombotic microangiopathy (grade 4) with thrombocytopenia and acute kidney injury. Three patients had glucocorticoid-related exacerbation (grade 3) of Danon disease-related skeletal myopathy. One patient with left ventricular systolic dysfunction at baseline had progressive heart failure and underwent transplantation 5 months after infusion. In the six patients with normal left ventricular ejection fraction at baseline, we observed cardiac LAMP2 protein expression and a reduction from baseline in or stabilization of the left ventricular mass index, preservation of left ventricular ejection fraction, and reduction in or stabilization of the levels of cardiac troponin I and N-terminal pro-B-type natriuretic peptide. At 24 to 54 months, all the patients were alive, with complete resolution of side effects. CONCLUSIONS:A single infusion of RP-A501 appeared to be safe and was associated with cardiac LAMP2 expression and evidence of clinical improvement over a period of 24 to 54 months. (Funded by Rocket Pharmaceuticals; ClinicalTrials.gov number, NCT03882437.).

Introduction Hereditary angioedema (HAE) is a rare genetic disorder marked by plasma kallikrein hyperactivity, causing subcutaneous and submucosal edema, with potentially life-threatening upper respiratory tract swelling. Current treatments require lifelong administration, with many patients still experiencing attacks. Our strategy seeks durable relief for HAE patients by employing a high-fidelity nuclease delivered via proprietary lipid nanoparticles (LNPs) to edit/inactivate the KLKB1 gene. Methods A proprietary gene editing system was used for KLKB1 editing. This system provides higher specificity over nucleases such as Cas9 via a dual-gRNA mechanism that spatially restricts activity. Human-specific gRNAs were evaluated in primary human hepatocytes (PHHs). Off-target editing was quantified by amplicon-seq. Editing efficiency was assessed in both humanized-liver TK-NOG mice and genetically humanized mice, while in vivo safety and impact on HAE biomarkers were tested in WT mice. A pilot study in non-human primates (NHP) evaluated preliminary tolerability and editing. Results KLKB1 editing in PHHs showed >90% efficiency with 85% reduction in protein secretion and very low off-target editing (<0.1%). In humanized mice, 60% KLKB1 editing was achieved with no off-target edits. In WT mice, dose-responsive editing resulted in >90% reduction in serum pre-kallikrein levels, with no serious adverse effects. In NHPs, we observed tolerability up to max doses tested and meaningful KLKB1 editing after single-dose administration. Conclusions P-KLKB1-101 offers high specificity via high-fidelity targeted gene editing while potentially enabling durable reduction in kallikrein activity. These preliminary data support further development for HAE, and the potential for other future high-fidelity genetic medicines.

Current first-line treatments for Hemophilia A patients are clotting factor replacement or bi-specific antibodies. These treatments require continuous, lifelong infusions, yet many patients have breakthrough bleeds. There remains a high unmet need for safe, effective, and durable therapies. We have developed a liver-directed non-viral in vivo gene insertion approach using the piggyBac® DNA insertion system. Unlike conventional AAV-based gene therapy, our platform enables delivery of large transgenes, the ability to stably and efficiently integrate the therapeutic transgene into the genome, and the potential for re-dosing to titrate to target FVIII activity levels. A key challenge for all gene insertion systems relates to the need to safely and efficiently deliver the transgene DNA. In the current study we evaluated a novel hepatocyte-targeted non-viral platform able to co-deliver both DNA and mRNA with superior safety and specificity relative to the traditional liver-directed LNP concept. We first evaluated a two nanoparticle system using conventional 4-component liver-directed LNPs, with one lipid nanoparticle (LNP) encapsulating the mRNA for the super piggyBac (SPB) transposase (LNP-SPB), and a second LNP encapsulating a plasmid containing the hFVIII transposon DNA (LNP-hFVIII). We subsequently developed a novel co-encapsulated LNP formulation, comprising both SPB mRNA and transposon plasmid DNA (LNP-SPB-hFVIII). In juvenile WT mice we observed a 50% increase in hFVIII antigen expression compared to the dual nanoparticle approach. This was further validated in a severe hemophilia A mouse model (FVIII knock-out). Following a single dose of the co-encapsulated LNP-SPB-hFVIII to adult hemophilia A mice tolerized to human FVIII, we observed ~30% of normal hFVIII expression sustained over the duration of the 7 month study. To further support the concept of re-dosing, we treated immunocompetent adult hemophilia A mice tolerized to human FVIII with repeated administrations every 3 weeks. We observed a dose-proportionate increase in FVIII activity after each administration, reaching an average hFVIII activity of 96% of normal, following 3 repeated doses. Traditional LNPs use several lipid components that typically include an ionizable lipid, cholesterol, a polyethylene glycol (PEG) lipid, and a structural lipid, each with a unique function to effectively encapsulate and deliver the DNA and mRNA required for the piggyBac platform. Cellular transfection of these traditional LNPs has been demonstrated to occur by passive mechanisms such as micropinocytosis or Apolipoprotein E (ApoE)-mediated endocytosis by the low-density lipoprotein receptor (LDLR). However, this can also result in delivery to unwanted cell-types, particularly tissue-resident immune cells. To address this potential failure mode, we explored the addition of a N-Acetylgalactosamine (GalNac)-based targeting ligand to our co-encapsulated LNP formulation (LNP-SPB-hFVIII-G). In immunocompetent animals, targeted LNPs yielded a ~2-3-log reduction in pro-inflammatory serum cytokines (IL-6, IFNɣ) while maintaining high hFVIII expression and no elevation in transaminases (ALT, AST). In conclusion, our results demonstrate the capabilities of the piggyBac DNA insertion system and non-viral approach in providing stable FVIII transgene expression through genomic integration, along with the potential for redosing. Additionally, we have highlighted the tolerability profile of our current generation of liver-targeted non-viral delivery platform. Altogether, these data provide proof-of-principle toward developing an effective and durable therapy for Hemophilia A.

Background Danon disease (DD) is a rare, X-linked monogenic cardiomyopathy caused by mutations in the LAMP2 gene which is essential for autophagy. In male patients, DD is characterized by a severe, progressive hypertrophic cardiomyopathy and arrhythmias resulting in median mortality under 20 years (y). This open-label Phase 1 trial in DD males evaluates systemic in vivo gene therapy (RP-A501) with adeno-associated virus 9 (AAV9) and a normal copy of the human LAMP2B gene. Pediatric evaluation was preceded by treatment of two adult DD cohorts with evidence of manageable safety, LAMP2B cardiac gene and protein expression and preliminary clinical efficacy. In this initial pediatric cohort, there was specific emphasis on minimization of immune responses, particularly complement activation and thrombotic microangiopathy (TMA) that have been associated with systemic AAV therapy in pediatric and adult evaluations in monogenic neuromuscular, metabolic and coagulation disorders. Methods An enhanced immunosuppressive regimen incorporating rituximab, sirolimus and limited corticosteroids was implemented to minimize the risk of complement-mediated events and steroid exposure after a single IV infusion of RP-A501 (AAV9.LAMP2B) at 6.7 × 1013 GC/kg with weight-based viral titer caps. Results Two pediatric patients (ages 11 and 12y) have received IV RP-A501 (6.7 × 1013 GC/kg). Platelet counts remained within normal range with modest decreases during days 6-14 post-infusion. Complement soluble membrane attack complex (sC5b-9) increased during days 5-14 post-infusion; no increases >200ng/mL over any 24 hour period were observed; peak values remained <450ng/mL (ULN 250 ng/mL) and were markedly lower than those observed in the adult cohort. Hemoglobin and creatinine levels remained stable during the initial weeks post-infusion. Transaminases and liver-focused parameters (GGT and bilirubin) were also largely stable. Limited and transient troponin increases were noted, consistent with known AAV9 cardiotropism. Increases in neutralizing and anti-capsid antibodies were observed and were more modest than those in the adult cohort. Managed with a steroid-sparing regimen, steroid induced exacerbations of DD-related skeletal myopathy in the pediatric patients were limited relative to those reported in the adult cohort. As previously reported, all adult patients with observed immunosuppressive regimen compliance (N=4) had evidence of cardiac LAMP2B expression within 6 months. Evaluation of gene expression in the pediatric patients is currently underway. Conclusions Robust immunomodulation during the initial days post-infusion enabled administration of this first-in-pediatric RP-A501 gene therapy for DD cardiomyopathy without evidence of immune-mediated clinical sequelae. Transient inhibition of humoral and cell-mediated immune response is intended to enable optimal RP-A501 cardiomyocyte delivery and transduction in order to arrest and potentially reverse the rapidly progressive cardiomyopathy associated with DD. Danon disease (DD) is a rare, X-linked monogenic cardiomyopathy caused by mutations in the LAMP2 gene which is essential for autophagy. In male patients, DD is characterized by a severe, progressive hypertrophic cardiomyopathy and arrhythmias resulting in median mortality under 20 years (y). This open-label Phase 1 trial in DD males evaluates systemic in vivo gene therapy (RP-A501) with adeno-associated virus 9 (AAV9) and a normal copy of the human LAMP2B gene. Pediatric evaluation was preceded by treatment of two adult DD cohorts with evidence of manageable safety, LAMP2B cardiac gene and protein expression and preliminary clinical efficacy. In this initial pediatric cohort, there was specific emphasis on minimization of immune responses, particularly complement activation and thrombotic microangiopathy (TMA) that have been associated with systemic AAV therapy in pediatric and adult evaluations in monogenic neuromuscular, metabolic and coagulation disorders. An enhanced immunosuppressive regimen incorporating rituximab, sirolimus and limited corticosteroids was implemented to minimize the risk of complement-mediated events and steroid exposure after a single IV infusion of RP-A501 (AAV9.LAMP2B) at 6.7 × 1013 GC/kg with weight-based viral titer caps. Two pediatric patients (ages 11 and 12y) have received IV RP-A501 (6.7 × 1013 GC/kg). Platelet counts remained within normal range with modest decreases during days 6-14 post-infusion. Complement soluble membrane attack complex (sC5b-9) increased during days 5-14 post-infusion; no increases >200ng/mL over any 24 hour period were observed; peak values remained <450ng/mL (ULN 250 ng/mL) and were markedly lower than those observed in the adult cohort. Hemoglobin and creatinine levels remained stable during the initial weeks post-infusion. Transaminases and liver-focused parameters (GGT and bilirubin) were also largely stable. Limited and transient troponin increases were noted, consistent with known AAV9 cardiotropism. Increases in neutralizing and anti-capsid antibodies were observed and were more modest than those in the adult cohort. Managed with a steroid-sparing regimen, steroid induced exacerbations of DD-related skeletal myopathy in the pediatric patients were limited relative to those reported in the adult cohort. As previously reported, all adult patients with observed immunosuppressive regimen compliance (N=4) had evidence of cardiac LAMP2B expression within 6 months. Evaluation of gene expression in the pediatric patients is currently underway. Robust immunomodulation during the initial days post-infusion enabled administration of this first-in-pediatric RP-A501 gene therapy for DD cardiomyopathy without evidence of immune-mediated clinical sequelae. Transient inhibition of humoral and cell-mediated immune response is intended to enable optimal RP-A501 cardiomyocyte delivery and transduction in order to arrest and potentially reverse the rapidly progressive cardiomyopathy associated with DD.

Cutting-edge gene editing holds enormous promise for tackling devastating genetic diseases like hereditary angioedema (HAE). Here, we describe the efficient inactivation of the gene encoding pre-kallikrein, KLKB1, using our proprietary Cas-CLOVER™ high-fidelity nuclease with our non-viral, lipid nanoparticle (LNP) delivery system. Genetic inactivation of KLKB1 is an alternative clinical approach that provides durable relief to both Type I and II HAE. HAE is a rare genetic disease characterized by subcutaneous and submucosal edema, with swelling of the upper respiratory tract posing a life-threatening situation. Type I and II HAE are the most common types and are caused by mutations in the SERPING1 gene, which leads to compromised production or function of the C1 protease inhibitor. Strategies for treatment and prophylaxis include the restoration of C1 inhibitor function, or downstream antagonism of active plasma kallikrein. Safe and effective gene editing of KLKB1 could be a viable alternative for patients not adequately responding to the current standard of care. However, gene editing approaches must demonstrate an exquisitely high level of fidelity for optimal safety. To demonstrate such an approach with Cas-CLOVER, multiple guide RNAs (gRNA) targeting the human KLKB1 gene were screened in human hepatoma cell lines to identify gRNA pairs with optimal editing. Next, we evaluated KLKB1 protein reduction in primary human hepatocytes (PHH) that were incubated with LNPs encapsulating Cas-CLOVER mRNA along with each gRNA pair. Lead candidate gRNAs showed robust KLKB1 editing in a dose-responsive manner, achieving >65% editing and >85% reduction in KLKB1 protein secreted into culture medium at 0.5 ug/mL (EC90). To evaluate Cas-CLOVER off-target activity, oligo incorporation by iGUIDE was carried out by a licensed contract research organization. In this assay, double-stranded oligodeoxyribonucleotides (dsODNs) were co-electroporated with Cas-CLOVER mRNA, along with our lead KLKB1 gRNA pair, in the Huh7 cell line, and candidate off-target sites were identified by Illumina next-generation sequencing. Off-target activity was assessed by amplicon-seq at the eight top sites nominated by iGUIDE. In PHHs treated with 0.5 ug/mL of Cas-CLOVER LNPs, off-target editing was detected in 3/8 sites at very low levels (<0.25%). Remarkably, this low level of off-target editing remained unchanged when PHHs were treated with 10-fold higher concentrations of Cas-CLOVER LNP. For further evaluation of our platform, we sought to determine KLKB1 editing efficiency and fidelity in a mouse model of liver humanization. TK-Nog mice engrafted with PHHs were treated with a single intravenous injection of an LNP formulation co-encapsulating Cas-CLOVER mRNA and our lead KLKB1 gRNA pair. Amplicon-seq analysis demonstrated that 60% of KLKB1 alleles in the liver were edited. Importantly, no off-target editing was detected among the top eight sites identified by iGUIDE, including the three off-target sites validated in cultured PHHs. Next, we evaluated efficacy and tissue specificity of our platform in wild type mice. C57BL/6 male and female mice were dosed with LNP encapsulating Cas-CLOVER mRNA and mouse Klkb1-targeting gRNAs. A single intravenous LNP injection achieved high Klkb1 editing (>50% of haploid genomes) in the liver and >80% reduction in serum pre-kallikrein levels. No Klkb1 editing was detected in gonads. In summary, these results highlight the efficacy and specificity of our high-fidelity Cas-CLOVER gene editing platform that enables targeted and therapeutically relevant kallikrein reduction in a fully non-viral manner. These data provide a promising foundation for the development of a highly specific gene editing therapy for HAE.

Background: Danon disease (DD) is an X-linked disorder caused by mutations in the LAMP2 gene that is essential for autophagy. DD is characterized by severe cardiomyopathy for which treatment options are limited. Median survival for male patients (pts) is approximately 19 years (y). DD natural history is notable for modest cardiac abnormalities in early phases detectable by measurement of left ventricular global longitudinal strain (GLS) or natriuretic peptides (NPs) followed by rapid progression, particularly in males during the second and third decade. Methods: This open-label Phase 1 study evaluates the safety of a single IV infusion of RP-A501 (AAV9. LAMP2B ) at dose levels of 6.7 x 10 13 GC/kg (low dose) and 1.1 x 10 14 GC/kg (high dose) in male DD pts in two age groups: ≥15 y and 8-14 y. Eligibility criteria include a DD diagnosis with LAMP2 mutation and cardiac involvement. Results: In Cohort 1 (N=3 pts, ≥15 y, 6.7 x 10 13 GC/kg), RP-A501 increased cardiac LAMP2B expression by Western blot (WB) and immunohistochemistry (IHC) in myocardial tissue. The two pts in Cohort 1 with observed immunosuppressive regimen compliance had high cardiac LAMP2B expression: 67.8% and 92.4% vs. normal control by IHC; up to 61% by WB. In both pts, NYHA Class improved from II to I. NPs were reduced by 59% and 42% at 9 and 12 months, respectively. All 3 pts had similar or mildly improved 6MWT, improved myocardial EM morphology with decreased autophagic vacuoles, improved/stabilized GLS and reported increases in physical activity. Adverse events were mostly manageable with transient immunosuppression. Reversible platelet decreases and transaminase elevations that returned to baseline within 2 months were seen. One patient treated with 1.1 x 10 14 GC/kg had complement-mediated thrombocytopenia and acute kidney injury requiring transient hemodialysis that fully recovered. An exacerbation of skeletal myopathy in 3 of the 5 patients resolved with steroid taper. Conclusions: This ongoing first-in-human trial demonstrates that the low dose of RP-A501 gene therapy for DD was generally well-tolerated, confers cardiac LAMP2B gene expression, and is associated with preliminary evidence of cardiac and clinical benefits.

Danon disease (DD) is a rare X-linked autophagic vacuolar myopathy associated with multiorgan dysfunction, including the heart, skeletal muscle, and liver. There are no specific treatments, and most male patients die from advanced heart failure during the second or third decade of life. DD is caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene, a key mediator of autophagy. LAMP2 has three isoforms: LAMP2A, LAMP2B, and LAMP2C. LAMP2B is the predominant isoform expressed in cardiomyocytes. This study evaluates the efficacy of human LAMP2B gene transfer using a recombinant adeno-associated virus 9 carrying human LAMP2B (AAV9.LAMP2B) in a Lamp2 knockout (KO) mouse, a DD model. AAV9.LAMP2B was intravenously injected into 2- and 6-month-old Lamp2 KO male mice to assess efficacy in adolescent and adult phenotypes. Lamp2 KO mice receiving AAV9.LAMP2B demonstrated dose-dependent restoration of human LAMP2B protein in the heart, liver, and skeletal muscle tissue. Impaired autophagic flux, evidenced by increased LC3-II, was abrogated by LAMP2B gene transfer in all tissues in both cohorts. Cardiac function was also improved, and transaminases were reduced in AAV9.LAMP2B-treated KO mice, indicating favorable effects on the heart and liver. Survival was also higher in the older cohort receiving high vector doses. No anti-LAMP2 antibodies were detected in mice that received AAV9.LAMP2B. In summary, LAMP2B gene transfer improves metabolic and physiologic function in a DD murine model, suggesting that a similar therapeutic approach may be effective for treating patients with this highly morbid disease.

Forkhead box O (FoxO) proteins and thyroid hormone (TH) have well established roles in cardiovascular morphogenesis and remodeling. However, specific role(s) of individual FoxO family members in stress-induced growth and remodeling of cardiomyocytes remains unknown. Here, we report that FoxO1, but not FoxO3, activity is essential for reciprocal regulation of types II and III iodothyronine deiodinases (Dio2 and Dio3, respectively), key enzymes involved in intracellular TH metabolism. We further show that Dio2 is a direct transcriptional target of FoxO1, and the FoxO1–Dio2 axis governs TH-induced hypertrophic growth of neonatal cardiomyocytes in vitro and in vivo. Utilizing transverse aortic constriction as a model of hemodynamic stress in wild-type and cardiomyocyte-restricted FoxO1 knockout mice, we unveil an essential role for the FoxO1–Dio2 axis in afterload-induced pathological cardiac remodeling and activation of TRα1. These findings demonstrate a previously unrecognized FoxO1–Dio2 signaling axis in stress-induced cardiomyocyte growth and remodeling and intracellular TH homeostasis.

AIMS:Considerable evidence points to critical roles of intracellular Ca2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca2+ homeostasis and autophagy.METHODS AND RESULTS:Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca2+ chelation using BAPTA-AM, whereas removal of extracellular Ca2+ had no effect, pointing to a role of intracellular Ca2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca2+-channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca2+. Furthermore, PC2 ablation was associated with impaired Ca2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca2+ stores. Finally, we provide evidence that Ca2+-mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types.CONCLUSION:Together, this study unveils PC2 as a novel regulator of autophagy acting through control of intracellular Ca2+ homeostasis.

Pancreatic β-cell dysfunction and death contribute to the onset of diabetes, and novel strategies of β-cell function and survival under diabetogenic conditions need to be explored. We previously demonstrated that Isx9, a small molecule based on the isoxazole scaffold, drives neuroendocrine phenotypes by increasing the expression of genes required for β-cell function and improves glycemia in a model of β cell regeneration. We further investigated the role of Isx9 in β-cell survival. We find that Isx9 drives the expression of Calbindin-D28K (D28K), a key regulator of calcium homeostasis, and plays a cytoprotective role through its calcium buffering capacity in β cells. Isx9 increased the activity of the calcineurin (CN)/cytoplasmic nuclear factor of the activated T-cells (NFAT) transcription factor, a key regulator of D28K, and improved the recruitment of NFATc1, cAMP response element-binding protein (CREB), and p300 to the D28K promoter. We found that nutrient stimulation increased D28K plasma membrane enrichment and modulated calcium channel activity in order to regulate glucose-induced insulin secretion. Isx9-mediated expression of D28K protected β cells against chronic stress induced by serum withdrawal or chronic inflammation by reducing caspase 3 activity. Consequently, Isx9 improved human islet function after transplantation in NOD-SCID mice in a streptozotocin-induced diabetes model. In summary, Isx9 significantly regulates expression of genes relevant to β cell survival and function, and may be an attractive therapy to treat diabetes and improve islet function post-transplantation.

Exercise has numerous beneficial metabolic effects. The central nervous system (CNS) is critical for regulating energy balance and coordinating whole body metabolism. However, a role for the CNS in the regulation of metabolism in the context of the exercise remains less clear. Here, using genetically engineered mice we assessed the requirement of steroidogenic factor-1 (SF-1) expression in neurons of the ventromedial hypothalamic nucleus (VMH) in mediating the beneficial effects of exercise on metabolism. We found that VMH-specific deletion of SF-1 blunts (a) the reductions in fat mass, (b) improvements in glycemia, and (c) increases in energy expenditure that are associated with exercise training. Unexpectedly, we found that SF-1 deletion in the VMH attenuates metabolic responses of skeletal muscle to exercise, including induction of PGC-1α expression. Collectively, this evidence suggests that SF-1 expression in VMH neurons is required for the beneficial effects of exercise on metabolism.

Background— L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress–induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein–coupled receptor–like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. Methods and Results— We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown–dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction–induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction–elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals Conclusion— PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein.

Division of Cardiology, Dept of Internal Medicine, UT Southwestern Medical Center, Dallas, TX; Advanced Center for Chronic Diseases (ACCDiS) & Centro de Estudios Moleculares de la Célula (CMEC), Facultad de Medicina & Facultad de Ciencias Químicas y Farmacéuticas, Santiago, Chile; Instituto de Ciencias Biomédicas, Facultad de Medicina; Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología, Universidad de Chile, Santiago, Chile; Dept of Cellular and Molecular Physiology; 6 Dept of Internal Medicine; Dept of Genetics, Yale University School of Medicine, New Haven, CT; Dept of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX

Background: Myosin regulatory light chain phosphorylation is necessary for normal cardiac performance. Results: Regulatory light chain phosphorylation is not affected by conditions affecting phosphorylation of other sarcomeric proteins, including β-adrenergic tone. Conclusion: Significant regulatory light chain phosphorylation in beating hearts is sustained physiologically by low cMLCK and MLCP activities. Significance: Constitutive regulatory light chain phosphorylation stabilizes cardiac performance. In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca2+ sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.

Cardiac myosin light chain kinase (cMLCK) phosphorylates a single site in the regulatory light chain (RLC) of myosin to increase Ca2+ sensitivity of myofilament contractions. A constellation of contractile protein phosphorylations in addition to RLC phosphorylation fine-tune actin-myosin myofibrillar force development to modulate cardiac performance. In the normal beating heart RLC is significantly phosphorylated (∼45%) which may play a constitutive physiological role to enhance cardiac performance. Conventional cMLCK knockout mice have dilated hearts with severely compromised cardiac performance at 10 weeks of age and older. To determine if the dilated phenotype caused by cMLCK knockout is preceded by a loss of cardiac performance associated with decreased phosphorylation of RLC and other myofibrillar proteins, we generated an acute model for the conditional knockout of cMLCK. We optimized the minimal amount of tamoxifen necessary for cMLCK ablation and assessed cardiac performance measured as fractional shortening by echocardiography. Hearts were then harvested for analyses of protein contents and phosphorylations. Five consecutive daily i.p. injections of 0.5 mg tamoxifen per mouse were sufficient to reduce cMLCK 80±2% by two weeks after the first tamoxifen injection. RLC phosphorylation was reduced to 15±2% while left ventricular internal diameter at end-diastole significantly increased by 1.4±0.3 mm and fractional shortening decreased 47±6%. There was no evidence of compensatory hypertrophy. Troponin-I and myosin binding protein-C phosphorylation at Ser23/24 and Ser282, respectively, did not significantly change. Both phosphorylations were reduced with propranolol treatment, which had no effect on RLC phosphorylation. These results suggest that constitutive RLC phosphorylation contributes to cardiac performance in the normal beating heart.