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Background: Left ventricular noncompaction cardiomyopathy (LVNC) was discovered half a century ago as a cardiomyopathy with excessive trabeculation and a thin ventricular wall. In the decades since, numerous studies have demonstrated that LVNC primarily has an effect on left ventricles (LVs) and is often associated with LV dilation and dysfunction. However, in part because of the lack of suitable mouse models that faithfully mirror the selective LV vulnerability in patients, mechanisms underlying the susceptibility of LVs to dilation and dysfunction in LVNC remain unknown. Genetic studies have revealed that deletions and mutations in PRDM16 (PR domain-containing 16) cause LVNC, but previous conditional Prdm16 knockout mouse models do not mirror the LVNC phenotype in patients, and the underlying molecular mechanisms by which PRDM16 deficiency causes LVNC are still unclear. Methods: Prdm16 cardiomyocyte–specific knockout ( Prdm16 cKO ) mice were generated and analyzed for cardiac phenotypes. RNA sequencing and chromatin immunoprecipitation deep sequencing were performed to identify direct transcriptional targets of PRDM16 in cardiomyocytes. Single-cell RNA sequencing in combination with spatial transcriptomics was used to determine cardiomyocyte identity at the single-cell level. Results: Cardiomyocyte-specific ablation of Prdm16 in mice caused LV-specific dilation and dysfunction, as well as biventricular noncompaction, which fully recapitulated LVNC in patients. PRDM16 functioned mechanistically as a compact myocardium-enriched transcription factor that activated compact myocardial genes while repressing trabecular myocardial genes in LV compact myocardium. Consequently, Prdm16 cKO LV compact myocardial cardiomyocytes shifted from their normal transcriptomic identity to a transcriptional signature resembling trabecular myocardial cardiomyocytes or neurons. Chamber-specific transcriptional regulation by PRDM16 was attributable in part to its cooperation with LV-enriched transcription factors Tbx5 and Hand1. Conclusions: These results demonstrate that disruption of proper specification of compact cardiomyocytes may play a key role in the pathogenesis of LVNC. They also shed light on underlying mechanisms of the LV-restricted transcriptional program governing LV chamber growth and maturation, providing a tangible explanation for the susceptibility of LV in a subset of LVNC cardiomyopathies.

Background Emery-Dreifuss muscular dystrophy, caused by mutations in genes such as emerin (EMD) or lamin A/C (LMNA), is a disorder affecting the joints, muscles, and heart, with a wide spectrum of patient phenotypes including muscle wasting and cardiac conduction defects. Methods and results Here we report a multi-generation family from the Hunan Province of China. Affected family members displayed an uncommon clinical presentation of serious cardiac conduction abnormalities at an early age and a high incidence of sudden cardiac death along with mild skeletal muscular atrophy and joint contracture. Clinical analysis of affected members provided evidence of X-linked recessive inheritance. Consequently, using Sanger sequencing of X chromosome exomes, we identified a novel duplication mutation (c.405dup/p.Asp136X) in the EMD gene as the cause for the disease in this family. This variant is a novel mutation that has not been previously reported in Pubmed, Clinvar or other cases reported in the Human Gene Mutation Database. Conclusion Our finding expands the mutation spectrum of Emery-Dreifuss muscular dystrophy and provides a rationale for EMD mutation testing in cases of X-linked inherited cardiac conduction disease and sudden cardiac death, even in those lacking pathognomonic neuromuscular features.

Objectives Recent studies revealed LRRC8A to be an essential component of volume‐regulated anion channel (VRAC), which regulates cellular volume homeostasis. However, evidence for the contribution of LRRC8A‐dependent VRAC activity in vascular smooth muscle cells (VSMCs) is still lacking, and the relevant functional role of LRRC8A in VSMCs remains unknown. The primary goal of this study was to elucidate the role of LRRC8A in VRAC activity in VSMCs and the functional role of LRRC8A in cerebrovascular remodeling during hypertension. Materials and Methods siRNA‐mediated knockdown and adenovirus‐mediated overexpression of LRRC8A were used to elucidate the electrophysiological properties of LRRC8A in basilar smooth muscle cells (BASMCs). A smooth muscle–specific overexpressing transgenic mouse model was used to investigate the functional role of LRRC8A in cerebrovascular remodeling. Results LRRC8A is essential for volume‐regulated chloride current (I Cl, Vol) in BASMCs. Overexpression of LRRC8A induced a voltage‐dependent Cl− current independently of hypotonic stimulation. LRRC8A regulated BASMCs proliferation through activation of WNK1/PI3K‐p85/AKT axis. Smooth muscle‐specific upregulation of LRRC8A aggravated Angiotensin II‐induced cerebrovascular remodeling in mice. Conclusions LRRC8A is an essential component of VRAC and is required for cell volume homeostasis during osmotic challenge in BASMCs. Smooth muscle specific overexpression of LRRC8A increases BASMCs proliferation and substantially aggravates basilar artery remodeling, revealing a potential therapeutic target for vascular remodeling in hypertension., The schematic diagram for LRRC8A role in cerebrovascular remodeling. LRRC8A is an essential component of VRAC in BASMCs. During the challenge of hypertension, the activated LRRC8A channel‐mediated‐Cl− efflux increases WNK1 phosphorylation, which in turn triggers AKT phosphorylation and promotes BASMCs proliferation, eventually exacerbates hypertension‐induced cerebrovascular vascular remodeling.

Nexilin (NEXN) was recently identified as a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules. Loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy (DCM) and premature death. A 3 bp deletion (1948–1950del) leading to loss of the glycine in position 650 (G650del) is classified as a variant of uncertain significance in humans and may function as an intermediate risk allele. To determine the effect of the G650del variant on cardiac structure and function, we generated a G645del-knockin (G645del is equivalent to human G650del) mouse model. Homozygous G645del mice express about 30% of the Nexn expressed by WT controls and exhibited a progressive DCM characterized by reduced T-tubule formation, with disorganization of the transverse-axial tubular system. On the other hand, heterozygous Nexn global KO mice and genetically engineered mice encoding a truncated Nexn missing the first N-terminal actin-binding domain exhibited normal cardiac function, despite expressing only 50% and 20% of the Nexn, respectively, expressed by WT controls, suggesting that not only quantity but also quality of Nexn is necessary for a proper function. These findings demonstrated that Nexn G645 is crucial for Nexn’s function in tubular system organization and normal cardiac function., A mouse model of a dilated cardiomyopathy associated mutation in Nexilin reveals roles in tubular system organization and normal cardiac function.

Background: NEXN (nexilin) is a protein of the junctional membrane complex required for development of cardiac T-tubules. Global and cardiomyocyte-specific loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy and premature death. Therefore, little is known as to the role of NEXN in adult cardiomyocytes. Transverse-axial tubular system remodeling are well-known features in heart failure. Although NEXN is required during development for T-tubule formation, its role, if any, in mature T-tubules remains to be addressed. Methods: Nexn inducible adult cardiomyocyte-specific KO mice were generated. Comprehensive morphological and functional analyses were performed. Heart samples (n>3) were analyzed by molecular, biochemical, and electron microscopy analyses. Isolated single adult cardiomyocytes were analyzed by confocal microscopy, and myocyte shortening/re-lengthening and Ca 2+ transient studies were conducted. Results: Inducible cardiomyocyte-specific loss of Nexn in adult mice resulted in a dilated cardiomyopathy with reduced cardiac function (13% reduction in percentage fractional shortening; P Nexn inducible adult cardiomyocyte-specific KO mice reduced by 40% with respect to controls, P Conclusions: Results here reported reveal NEXN to be a pivotal component of adult junctional membrane complexes required for maintenance of transverse-axial tubular architecture. These results demonstrate that NEXN plays an essential role in the adult cardiomyocyte and give further understanding of pathological mechanisms responsible for cardiomyopathy in patients carrying mutations in the NEXN gene.

Summary: To facilitate understanding of human cardiomyocyte (CM) subtype specification, and the study of ventricular CM biology in particular, we developed a broadly applicable strategy for enrichment of ventricular cardiomyocytes (VCMs) derived from human embryonic stem cells (hESCs). A bacterial artificial chromosome transgenic H9 hESC line in which GFP expression was driven by the human ventricular-specific myosin light chain 2 (MYL2) promoter was generated, and screened to identify cell-surface markers specific for MYL2-GFP-expressing VCMs. A CD77+/CD200− cell-surface signature facilitated isolation of >97% cardiac troponin I-positive cells from H9 hESC differentiation cultures, with 65% expressing MYL2-GFP. This study provides a tool for VCM enrichment when using some, but not all, human pluripotent stem cell lines. Tools generated in this study can be utilized toward understanding CM subtype specification, and enriching for VCMs for therapeutic applications. : In this article, Evans and colleagues generated an H9 BAC transgenic reporter cell line and performed a flow cytometry screen to identify a cell-surface signature specific for MYL2-GFP-expressing VCMs. The cell-surface signature, CD77+/CD200−, facilitated isolation of a nearly pure hESC-derived CM population, enriched for VCMs. VCM enrichment was achieved when using some, but not all, human pluripotent stem cell lines. Tools generated in this study serve to advance our understanding of CM subtype specification, commitment, and maturation. Keywords: cardiac differentiation, human embryonic stem cells, ventricular cardiomyocytes, cell-surface marker signature

Background: Membrane contact sites are fundamental for transmission and translation of signals in multicellular organisms. The junctional membrane complexes in the cardiac dyads, where transverse (T) tubules are juxtaposed to the sarcoplasmic reticulum, are a prime example. T-tubule uncoupling and remodeling are well-known features of cardiac disease and heart failure. Even subtle alterations in the association between T-tubules and the junctional sarcoplasmic reticulum can cause serious cardiac disorders. NEXN (nexilin) has been identified as an actin-binding protein, and multiple mutations in the NEXN gene are associated with cardiac diseases, but the precise role of NEXN in heart function and disease is still unknown. Methods: Nexn global and cardiomyocyte-specific knockout mice were generated. Comprehensive phenotypic and RNA sequencing and mass spectrometry analyses were performed. Heart tissue samples and isolated single cardiomyocytes were analyzed by electron and confocal microscopy. Results: Global and cardiomyocyte-specific loss of Nexn in mice resulted in a rapidly progressive dilated cardiomyopathy. In vivo and in vitro analyses revealed that NEXN interacted with junctional sarcoplasmic reticulum proteins, was essential for optimal calcium transients, and was required for initiation of T-tubule invagination and formation. Conclusions: These results demonstrated that NEXN is a pivotal component of the junctional membrane complex and is required for initiation and formation of T-tubules, thus providing insight into mechanisms underlying cardiomyopathy in patients with mutations in NEXN .

Ca2+ signaling is important for many cellular and physiological processes, including cardiac function. Although sarcoplasmic reticulum (SR) proteins involved in Ca2+ signaling have been shown to be phosphorylated, the biochemical and physiological roles of protein phosphorylation within the lumen of the SR remain essentially uncharacterized. Our laboratory recently identified an atypical protein kinase, Fam20C, which is uniquely localized to the secretory pathway lumen. Here, we show that Fam20C phosphorylates several SR proteins involved in Ca2+ signaling, including calsequestrin2 and Stim1, whose biochemical activities are dramatically regulated by Fam20C mediated phosphorylation. Notably, phosphorylation of Stim1 by Fam20C enhances Stim1 activation and store-operated Ca2+ entry. Physiologically, mice with Fam20c ablated in cardiomyocytes develop heart failure following either aging or induced pressure overload. We extended these observations to show that non-muscle cells lacking Fam20C display altered ER Ca2+ signaling. Overall, we show that Fam20C plays an overarching role in ER/SR Ca2+ homeostasis and cardiac pathophysiology.

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2–associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

Defective protein quality control (PQC) systems are implicated in multiple diseases, with molecular chaperones/co-chaperones being critical to PQC. Cardiomyocytes are constantly challenged by mechanical and metabolic stress, placing great demand on the PQC system. Mutations and downregulation of the co-chaperone protein B cl-2- a ssociated athano g ene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to Dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and mechanisms by which the E455K mutation lead to DCM remained obscure. Here, we found that cardiac-specific BAG3 knockout (CKO) and cardiac-specific E455K BAG3 knockin mice developed DCM. Comparable phenotypes in the two mutants demonstrated that the E455K mutation resulted in loss-of-function, and experiments revealed that the E455K mutation disrupted interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a specific subset of proteins required for metabolic and contractile function of cardiomyocytes was enriched in the insoluble fraction. Together, these observations suggested that interaction between BAG3 and HSP70 was essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide new insight into the pathogenesis of heart failure caused by defects in BAG3 pathways, suggesting that increasing protein levels of BAG3 may be of therapeutic benefit in heart failure.