Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/−) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/− hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure

PPM1K, a novel regulator of metabolism and autophagy in the heart

The general aim of this work was to understand the relationship between mitochondria and autophagy, with a focus on heart biology. Autophagy is of extreme importance for cardiac function. Animal models of impaired autophagy display cardiac phenotypes at basal levels as well as when stressed. In this work we dissected molecularly two different faces of mitochondrial biology related to autophagy: BCAA-catabolism mediated regulation of mTORC1, and the role of the deubiquitinating enzyme USP8, involved in EGFR signaling and mitophagy, in heart mitochondrial function and more generally in heart function. Tissues can adapt to availability of different substrates by activating specific catabolic pathways that in most instances converge on mitochondria. Yet, how fluxes of metabolites through these organelles affect cellular processes is unclear. Here we show that PPM1K, a mitochondrial matrix protein phosphatase that controls the rate limiting step of branched chain amino acid (BCAA) catabolism, modulates mTORC1 activation and autophagy. PPM1K levels directly correlated with increased autophagy and reciprocally, PPM1K was induced upon starvation in vitro and in vivo, including in tissues where BCAA catabolism is considered marginal, like heart. Steady state metabolomics of labeled Leucine metabolites revealed that in the absence of PPM1K, TCA cycle intermediates were as expected decreased, whereas Leucine, its ketoisocaproic ketoacid and surprisingly methionine were increased, potentially explaining the mTORC1 dependent autophagy inhibition. Our data suggest how mitochondrial BCAA catabolism can be sensed by mTORC1 to modulate autophagy. Activating mutations in the USP8 gene, coding for ubiquitin-specific protease 8, a deubiquitinase involved in endocytic trafficking and mitophagy, can cause Cushing’s syndrome. Usp8 inhibitors are therefore scrutinized to treat Cushing’s pituitary adenomas. However, because heart function requires mitophagy, it is unclear if Usp8 inhibitors could be detrimental for the already failing hearts of Cushing’s patients. Here we show that acute Usp8 genetic ablation in the mouse heart impairs mitochondrial function and autophagic clearance. Myocardial Usp8 deletion in adult mice resulted in cardiomyopathy associated with the accumulation of damaged and dysfunctional mitochondria. Mechanistically, we found that USP8 interacted with, and stabilized PINK1 that senses dysfunctional mitochondria and activates Parkin dependent mitophagy. Consequently, in cardiomyocytes and cells lacking USP8, PINK1 was not stabilized upon mitochondrial dysfunction, mitophagy was not activated in response to mitochondrial depolarization and chemical mitochondrial uncouplers led to cell death. Our data not only shed light on the mechanisms of mitophagy regulation, but also recommend caution in investigative anti Usp8 therapy for Cushing’s syndrome.The general aim of this work was to understand the relationship between mitochondria and autophagy, with a focus on heart biology. Autophagy is of extreme importance for cardiac function. Animal models of impaired autophagy display cardiac phenotypes at basal levels as well as when stressed. In this work we dissected molecularly two different faces of mitochondrial biology related to autophagy: BCAA-catabolism mediated regulation of mTORC1, and the role of the deubiquitinating enzyme USP8, involved in EGFR signaling and mitophagy, in heart mitochondrial function and more generally in heart function. Tissues can adapt to availability of different substrates by activating specific catabolic pathways that in most instances converge on mitochondria. Yet, how fluxes of metabolites through these organelles affect cellular processes is unclear. Here we show that PPM1K, a mitochondrial matrix protein phosphatase that controls the rate limiting step of branched chain amino acid (BCAA) catabolism, modulates mTORC1 activation and autophagy. PPM1K levels directly correlated with increased autophagy and reciprocally, PPM1K was induced upon starvation in vitro and in vivo, including in tissues where BCAA catabolism is considered marginal, like heart. Steady state metabolomics of labeled Leucine metabolites revealed that in the absence of PPM1K, TCA cycle intermediates were as expected decreased, whereas Leucine, its ketoisocaproic ketoacid and surprisingly methionine were increased, potentially explaining the mTORC1 dependent autophagy inhibition. Our data suggest how mitochondrial BCAA catabolism can be sensed by mTORC1 to modulate autophagy. Activating mutations in the USP8 gene, coding for ubiquitin-specific protease 8, a deubiquitinase involved in endocytic trafficking and mitophagy, can cause Cushing’s syndrome. Usp8 inhibitors are therefore scrutinized to treat Cushing’s pituitary adenomas. However, because heart function requires mitophagy, it is unclear if Usp8 inhibitors could be detrimental for the already failing hearts of Cushing’s patients. Here we show that acute Usp8 genetic ablation in the mouse heart impairs mitochondrial function and autophagic clearance. Myocardial Usp8 deletion in adult mice resulted in cardiomyopathy associated with the accumulation of damaged and dysfunctional mitochondria. Mechanistically, we found that USP8 interacted with, and stabilized PINK1 that senses dysfunctional mitochondria and activates Parkin dependent mitophagy. Consequently, in cardiomyocytes and cells lacking USP8, PINK1 was not stabilized upon mitochondrial dysfunction, mitophagy was not activated in response to mitochondrial depolarization and chemical mitochondrial uncouplers led to cell death. Our data not only shed light on the mechanisms of mitophagy regulation, but also recommend caution in investigative anti Usp8 therapy for Cushing’s syndrome

Compartmentation of cGMP Signaling in Induced Pluripotent Stem Cell Derived Cardiomyocytes during Prolonged Culture

The therapeutic benefit of stimulating the cGMP pathway as a form of treatment to combat heart failure, as well as other fibrotic pathologies, has become well established. However, the development and signal compartmentation of this crucial pathway has so far been overlooked. We studied how the three main cGMP pathways, namely, nitric oxide (NO)-cGMP, natriuretic peptide (NP)-cGMP, and β3-adrenoreceptor (AR)-cGMP, mature over time in culture during cardiomyocyte differentiation from human pluripotent stem cells (hPSC-CMs). After introducing a cGMP sensor for Förster Resonance Energy Transfer (FRET) microscopy, we used selective phosphodiesterase (PDE) inhibition to reveal cGMP signal compartmentation in hPSC-CMs at various times of culture. Methyl-β-cyclodextrin was employed to remove cholesterol and thus to destroy caveolae in these cells, where physical cGMP signaling compartmentalization is known to occur in adult cardiomyocytes. We identified PDE3 as regulator of both the NO-cGMP and NP-cGMP pathway in the early stages of culture. At the late stage, the role of the NO-cGMP pathway diminished, and it was predominantly regulated by PDE1, PDE2, and PDE5. The NP-cGMP pathway shows unrestricted locally and unregulated cGMP signaling. Lastly, we observed that maturation of the β3-AR-cGMP pathway in prolonged cultures of hPSC-CMs depends on the accumulation of caveolae. Overall, this study highlighted the importance of structural development for the necessary compartmentation of the cGMP pathway in maturing hPSC-CMs

Comprehensive autophagy evaluation in cardiac disease models

International audienceAutophagy is a highly conserved recycling mechanism essential for maintaining cellular homeostasis. The pathophysiological role of autophagy has been explored since its discovery 50 years ago, but interest in autophagy has grown exponentially over the last years. Many researchers around the globe have found that autophagy is a critical pathway involved in the pathogenesis of cardiac diseases. Several groups have created novel and powerful tools for gaining deeper insights into the role of autophagy in the aetiology and development of pathologies affecting the heart. Here, we discuss how established and emerging methods to study autophagy can be used to unravel the precise function of this central recycling mechanism in the cardiac system

Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes.

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/-) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure