Desmin is the major protein component of the intermediate filaments (IFs) cytoskeleton in muscle cells, including cardiac. The accumulation of cleaved and misfolded desmin is a cellular hallmark of heart failure (HF). These desmin alterations are reversed by therapy, suggesting a causal role for the IFs in the development of HF. Though IFs are known to play a role in the protection from stress, a mechanistic model of how that occurs is currently lacking. On the other hand, the heart is uniquely suited to study the function of the IFs, due to its inherent, cyclic contraction. That is, HF can be used as a model to address how IFs afford protection from mechanical, and possibly redox, stress. In this review we provide a brief summary of the current views on the function of the IFs, focusing on desmin. We also propose a new model according to which the propensity of desmin to aggregate may have been selected during evolution as a way to dissipate excessive mechanical and possibly redox stress. According to this model, though desmin misfolding may afford protection from acute injury, the sustained or excessive accumulation of desmin aggregates could impair proteostasis and contribute to disease.

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.

Variants in CRYAB can lead to desmin-related (cardio-)myopathy (DRM), a genetic muscle disorder with no curative treatment available. We introduced a homozygous CRYAB c.358G > A (p.Arg120Gly) mutation, which is established for the study of DRM in mice, into a donor human induced pluripotent stem cell (hiPSC) line. Control and mutant hiPSCs were tested for karyotype integrity and pluripotency marker expression. HiPSCs could be differentiated into endoderm, ectoderm and cardiomyocytes as a mesodermal derivative in vitro. CRYABhom hiPSC-derived cardiomyocytes developed intracellular CRYAB aggregates, which is a hallmark of DRM. This newly created mutant can be utilized to study DRM and cardiac proteinopathy in a human context.

Hypertrophic cardiomyopathy (HCM) is the most common myocardial genetic disease characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction with preserved or elevated ejection fraction. Thirty-five years after the identification of the first genetic variant in myosin heavy chain 7, other variants have been discovered in numerous components of the sarcomere, pointing to a primary defect in cardiomyocyte contractility. Still, a large portion of HCM patients does not have a pathogenic variant and others present with LVH of another genetic origin. Research has uncovered a primary driver of hypercontractility at the sarcomere level and diverse molecular and cellular mechanisms contributing to HCM, including alterations of calcium handling and proteolysis, microtubule modifications, energy deficiency, and the impact of noncardiomyocyte cell types. These discoveries have fueled preclinical and translational research, leading to the development of myosin inhibitors, which are now on the market, and gene-based therapeutic products. This review summarizes current knowledge on the genetics, mechanisms, and targeted treatments of HCM.

Rationale: Protein accumulation is a hallmark of many neurodegenerative and muscular diseases. Desmin-related (cardio-) myopathy (DRM), a well-studied model for cardiac muscle protein accumulation, is an autosomal dominant-inherited disease presenting with progressive muscle weakness, reduced quality of life, and shortened life span. To date, DRM patients are treated symptomatically and there is no causal treatment available. Independent of the genetic cause, most DRM patients display intracellular accumulation of desmin and its chaperone αB-crystallin (CRYAB). We previously conducted an unbiased high-throughput screen to identify novel effectors that reduce cardiomyocyte aggregate levels and found that downregulation of Janus kinase 1 (JAK1) resulted in lower aggregate load in neonatal mouse cardiomyocytes. Objective: In this study, we tested if the approved JAK inhibitor ruxolitinib ameliorates the disease phenotype in rodent and human CRYAB p.Arg120Gly DRM models. Methods and Results: We found that the mRNA levels of Jak1 and Stat3 were higher than any other JAK-signal transducer and activator of transcription (STAT) family members in neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The approved JAK1/2 inhibitor ruxolitinib and the JAK1 inhibitors solcitinib, upadacitinib, and filgotinib prevented accumulation of and cleared pre-existing CRYAB p.Arg120Gly protein aggregates in NRVMs and hiPSC-CMs. Importantly, the knockdown of Jak1 and Stat3 , but not Jak2 resulted in fewer aggregates. Moreover, ruxolitinib, Jak1 or Stat3 siRNA treatment enhanced the ubiquitin-proteasome system (UPS)-mediated degradation. Blocking UPS function blunted the effect of ruxolitinib or Jak1 siRNA on CRYAB p.Arg120Gly accumulation. RNAseq of NRVMs treated with Jak1 siRNA extracts revealed higher gene expression of important muscle E3 ubiquitinating enzymes. Knockdown of the E3 ligase Asb2 (Ankyrin Repeat And SOCS Box Protein 2) abolished the effect of ruxolitinib on CRYAB p.Arg120Gly aggregates. In DRM mice, phospho-STAT3 levels were markedly higher than in non-transgenic (NTG) mice with age. Ruxolitinib treatment or Jak1 knockout prevented cardiac dysfunction and reduced CRYAB p.Arg120Gly aggregate load in DRM mice. Conclusion: In this study, we uncovered the previously unknown effect of the approved drug ruxolitinib to enhance UPS-mediated degradation and prevent protein aggregates in cardiomyocytes.

BACKGROUND: Dilated cardiomyopathy caused by LMNA mutations is a severe cardiac condition marked by arrhythmias, contractile dysfunction, and excessive myocardial fibrosis, which collectively impair left ventricular function and increase the risk of heart failure. Although the disease has been well characterized, a lack of insight into the pathogenesis has impeded the development of therapies. METHODS: Here, we employed human induced pluripotent stem cells (hiPSCs) derived from a patient carrying a LMNA point mutation (c.665A>C, p.His222Pro), alongside a murine model carrying the same mutation, to investigate the functional and molecular abnormalities driving dilated cardiomyopathy. RESULTS: We demonstrated that LMNA patient-derived cardiomyocytes and engineered heart tissues exhibited elevated diastolic calcium levels and reduced sensitivity to external calcium, respectively, as well as hypocontractility. These cells also displayed nuclear shape abnormalities in 2-dimensional and 3-dimensional, a hallmark of LMNA -associated dilated cardiomyopathy, associated with disrupted chromosome spatial organization and altered gene expression profiles. Transcriptomic analysis revealed dysregulation of extracellular matrix remodeling and significant upregulation of Loxl2 in mutated hiPSC–cardiomyocytes, hiPSC–engineered heart tissues, and mice. Treatment with Simtuzumab, a Loxl2 inhibitor, effectively prevented cardiac dysfunction and fibrosis in vivo. CONCLUSIONS: Taken together, our findings underscore the crucial role of Loxl2 as a therapeutic target and suggest that its inhibition could be a promising strategy to preserve cardiac function in LMNA -associated dilated cardiomyopathy.