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.

Generation of left ventricle-like cardiomyocytes with improved structural, functional, and metabolic maturity from human pluripotent stem cells.

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV cardiomyocytes are, therefore, a potentially valuable therapeutical target. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are neither homogeneous nor functionally mature, which reduces their utility. Here, we exploit cardiac development knowledge to instruct differentiation of hPSCs specifically toward LV cardiomyocytes. Correct mesoderm patterning and retinoic acid pathway blocking are essential to generate near-homogenous LV-specific hPSC-CMs (hPSC-LV-CMs). These cells transit via first heart field progenitors and display typical ventricular action potentials. Importantly, hPSC-LV-CMs exhibit increased metabolism, reduced proliferation, and improved cytoarchitecture and functional maturity compared with age-matched cardiomyocytes generated using the standard WNT-ON/WNT-OFF protocol. Similarly, engineered heart tissues made from hPSC-LV-CMs are better organized, produce higher force, and beat more slowly but can be paced to physiological levels. Together, we show that functionally matured hPSC-LV-CMs can be obtained rapidly without exposure to current maturation regimes.

Dysfunctional mitochondria accumulate in a skeletal muscle knockout model of Smn1, the causal gene of spinal muscular atrophy.

The approved gene therapies for spinal muscular atrophy (SMA), caused by loss of survival motor neuron 1 (SMN1), greatly ameliorate SMA natural history but are not curative. These therapies primarily target motor neurons, but SMN1 loss has detrimental effects beyond motor neurons and especially in muscle. Here we show that SMN loss in mouse skeletal muscle leads to accumulation of dysfunctional mitochondria. Expression profiling of single myofibers from a muscle specific Smn1 knockout mouse model revealed down-regulation of mitochondrial and lysosomal genes. Albeit levels of proteins that mark mitochondria for mitophagy were increased, morphologically deranged mitochondria with impaired complex I and IV activity and respiration and that produced excess reactive oxygen species accumulated in Smn1 knockout muscles, because of the lysosomal dysfunction highlighted by the transcriptional profiling. Amniotic fluid stem cells transplantation that corrects the SMN knockout mouse myopathic phenotype restored mitochondrial morphology and expression of mitochondrial genes. Thus, targeting muscle mitochondrial dysfunction in SMA may complement the current gene therapy.

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 ß3-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-ß-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 ß3-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.

Autophagy 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.