BACKGROUND: Cardiac transverse tubules (T-tubules) are anchored to sarcomeric Z-discs by costameres to establish a regular spaced pattern. One of the major components of costameres is the dystrophin-glycoprotein complex (DGC). Nevertheless, how the assembly of the DGC coordinates with the formation and maintenance of T-tubules under physiological and pathological conditions remains unclear. METHODS: Given the known role of Ptpn23 (protein tyrosine phosphatase, nonreceptor type 23) in regulating membrane deformation, its expression in patients with dilated cardiomyopathy was determined. Taking advantage of Cre/Loxp, CRISPR/Cas9, and adeno-associated virus 9 (AAV9)–mediated in vivo gene editing, we generated cardiomyocyte-specific Ptpn23 and Actn2 (α-actinin-2, a major component of Z-discs) knockout mice. We also perturbed the DGC by using dystrophin global knockout mice ( Dmd ^E4* ). MM 4-64 and Di-8-ANEPPS staining, Cav3 immunofluorescence, and transmission electron microscopy were performed to determine T-tubule structure in isolated cells and intact hearts. In addition, the assembly of the DGC with Ptpn23 and dystrophin loss of function was determined by glycerol-gradient fractionation and SDS-PAGE analysis. RESULTS: The expression level of Ptpn23 was reduced in failing hearts from dilated cardiomyopathy patients and mice. Genetic deletion of Ptpn23 resulted in disorganized T-tubules with enlarged diameters and progressive dilated cardiomyopathy without affecting sarcomere organization. AAV9-mediated mosaic somatic mutagenesis further indicated a cell-autonomous role of Ptpn23 in regulating T-tubule formation. Genetic and biochemical analyses showed that Ptpn23 was essential for the integrity of costameres, which anchor the T-tubule membrane to Z-discs, through interactions with α-actinin and dystrophin. Deletion of α-actinin altered the subcellular localization of Ptpn23 and DGCs. In addition, genetic inactivation of dystrophin caused similar T-tubule defects to Ptpn23 loss-of-function without affecting Ptpn23 localization at Z-discs. Last, inducible Ptpn23 knockout at 1 month of age showed Ptpn23 is also required for the maintenance of T-tubules in adult cardiomyocytes. CONCLUSIONS: Ptpn23 is essential for cardiac T-tubule formation and maintenance along Z-discs. During postnatal heart development, Ptpn23 interacts with sarcomeric α-actinin and coordinates the assembly of the DGC at costameres to sculpt T-tubule spatial patterning and morphology.

Heart disease is the leading cause of mortality worldwide. Due to the limited proliferation rate of mature cardiomyocytes, adult mammalian hearts are unable to regenerate damaged cardiac muscle following injury. Instead, injured area is replaced by fibrotic scar tissue, which may lead to irreversible cardiac remodeling and organ failure. In contrast, adult zebrafish and neonatal mammalian possess the capacity for heart regeneration and have been widely used as experimental models. Recent studies have shown that multiple types of cells within the heart can respond to injury with the activation of distinct signaling pathways. Determining the specific contributions of each cell type is essential for our understanding of the regeneration network organization throughout the heart. In this review, we provide an overview of the distinct functions and coordinated cell behaviors of several major cell types including cardiomyocytes, endocardial cells, epicardial cells, fibroblasts, and immune cells. The topic focuses on their specific responses and cellular plasticity after injury, and potential therapeutic applications.

The histidine-rich calcium binding protein (HRC) Ser96Ala polymorphism was shown to correlate with ventricular arrhythmias and sudden death only in dilated cardiomyopathy patients but not in healthy human carriers. In the present study, we assessed the molecular and cellular mechanisms underlying human arrhythmias by adenoviral expression of the human wild-type (HRC^WT) or mutant HRC (HRC^S96A) in adult rat ventricular cardiomyocytes. Total HRC protein was increased by ∼50% in both HRC^WT- and HRC^S96A-infected cells. The HRC^S96A mutant exacerbated the inhibitory effects of HRC^WT on the amplitude of Ca²⁺ transients, prolongation of Ca²⁺ decay time, and caffeine-induced sarcoplasmic reticulum Ca²⁺ release. Consistent with these findings, HRC^S96A reduced maximal sarcoplasmic reticulum calcium uptake rate to a higher extent than HRC^WT. Furthermore, the frequency of spontaneous Ca²⁺ sparks, which was reduced by HRC^WT, was increased by mutant HRC^S96A under resting conditions although there were no spontaneous Ca²⁺ waves under stress conditions. However, expression of the HRC^S96A genetic variant in cardiomyocytes from a rat model of postmyocardial infarction heart failure induced dramatic disturbances of rhythmic Ca²⁺ transients. These findings indicate that the HRC Ser96Ala variant increases the propensity of arrhythmogenic Ca²⁺ waves in the stressed failing heart, suggesting a link between this genetic variant and life-threatening ventricular arrhythmias in human carriers.

Autophagy has stabilizing functions for cardiomyocytes. Recent studies indicate that an impairment in the autophagy pathway can seriously affect morphology and function, potentially leading to heart failure. However, the role and the underlying mechanism of the endosomal sorting complex required for transport (ESCRT) family protein, in particular the AAA-ATPase vacuolar protein sorting 4a (Vps4a), in regulating myocardial autophagy remains unclear. In the present study, cardiomyocyte-specific Vps4a knockout mice were generated by crossing Vps4a^flox/flox (Vps4a^fl/fl) with Myh6-cre transgenic mice. As a result, we observed a partially dilated left ventricular (LV) chamber, a significant increase in heart weight to body weight ratio (HW/BW), and heart weight to tibial length ratio (HW/TL), hypertrophic cardiomyopathy and early lethality starting at 3 months of age. Hematoxylin-eosin (HE), immunofluorescence assay (IFA), and Western blot (WB) revealed autophagosome accumulation in cardiomyocytes. A transcriptome-based analysis and autophagic flux tracking by AAV-RFP-GFP-LC3 showed that the autophagic flux was blocked in Vps4a knockout cardiomyocytes. In addition, we provided in vitro evidence demonstrating that Vps4a and LC3 were partially co-localized in cardiomyocytes, and the knockdown of Vps4a led to the accumulation of autophagosomes in cardiomyocytes. Similarly, the transfection of cardiomyocytes with adenovirus (Adv) mCherry-GFP-LC3 further indicated that the autophagic flux was blocked in cells with deficient levels of Vps4a. Finally, an electron microscope (EM) showed that the compromised sealing of autophagosome blocked the autophagic flux in Vps4a-depleted cardiomyocytes. These findings revealed that Vps4a contributed to the sealing of autophagosomes in cardiomyocytes. Therefore, we demonstrated that Vps4a deletion could block the autophagic flux, leading to the accumulation of degradation substances and compromised cardiac function. Overall, this study provides insights into a new theoretical basis for which autophagy may represent a therapeutic target for cardiovascular diseases.

The myocardial wall arises from a single layer of cardiomyocytes, some delaminate to create trabeculae while others remain in the compact layer. However, the mechanisms governing cardiomyocyte fate decisions remain unclear. Using single-cell RNA sequencing, genetically encoded biosensors, and in toto live imaging, we observe intrinsic variations in erbb2 expression and its association with trabecular fate. Specifically, erbb2 promotes PI3K activity and recruits the Arp2/3 complex, inducing a polarized accumulation of the actomyosin network to drive cell delamination. Subsequently, the lineage-committed nascent trabeculae trigger Notch activity in neighboring cardiomyocytes to suppress erbb2 expression and reduce cell tension, thereby confining them to the compact layer. Overall, this genetic and cellular interplay governs compact and trabecular cell fate determination to orchestrate myocardial pattern formation.