Yongxin Mu

Normalization of Naxos plakoglobin levels restores cardiac function in mice

Arrhythmogenic cardiomyopathy (AC) is associated with mutations in genes encoding intercalated disc proteins and ultimately results in sudden cardiac death. A subset of patients with AC have the autosomal recessive cardiocutaneous disorder Naxos disease, which is caused by a 2-base pair deletion in the plakoglobin-encoding gene JUP that results in a truncated protein with reduced expression. In mice, cardiomyocyte-specific plakoglobin deficiency recapitulates many aspects of human AC, and overexpression of the truncated Naxos-associated plakoglobin also results in an AC-like phenotype; therefore, it is unclear whether Naxos disease results from loss or gain of function consequent to the plakoglobin mutation. Here, we generated 2 knockin mouse models in which endogenous Jup was engineered to express the Naxos-associated form of plakoglobin. In one model, Naxos plakoglobin bypassed the nonsense-mediated mRNA decay pathway, resulting in normal levels of the truncated plakoglobin. Moreover, restoration of Naxos plakoglobin to WT levels resulted in normal heart function. Together, these data indicate that a gain of function in the truncated form of the protein does not underlie the clinical phenotype of patients with Naxos disease and instead suggest that insufficiency of the truncated Naxos plakoglobin accounts for disease manifestation. Moreover, these results suggest that increasing levels of truncated or WT plakoglobin has potential as a therapeutic approach to Naxos disease.

Cypher/ZASP is a novel A-kinase anchoring protein.

Background: Cypher/ZASP plays an essential structural role in cardiac muscle. Results: Cypher/ZASP specifically interacted with PKARIIα and calcineurin. Conclusion: Cypher/ZASP is a novel AKAP acting as a sarcomeric signaling center for potential phosphorylation regulation the function of channels and myofilament proteins. Significance: Cypher/ZASP-PKA-calcineurin complex expands our understanding the role of Cypher/ZASP in the heart. PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser265 and Ser296 by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser1928 induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy

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.

No Contribution of IP3-R(2) to Disease Phenotype in Models of Dilated Cardiomyopathy or Pressure Overload Hypertrophy

Background—We investigated the contribution of inositol(1,4,5)-trisphosphate (Ins(1,4,5)P3 [IP3]) receptors (IP3-R) to disease progression in mouse models of dilated cardiomyopathy (DCM) and pressure overload hypertrophy. Mice expressing mammalian sterile 20–like kinase and dominant-negative phosphatidylinositol-3-kinase in heart (Mst1×dn-PI3K-2Tg; DCM-2Tg) develop severe DCM and conduction block, associated with increased expression of type 2 IP3-R (IP3-R(2)) and heightened generation of Ins(1,4,5)P3. Similar increases in Ins(1,4,5)P3 and IP3-R(2) are caused by transverse aortic constriction. Methods and Results—To evaluate the contribution of IP3-R(2) to disease progression, the DCM-2Tg mice were further crossed with mice in which the type 2 IP3-R (IP3-R(2)−/−) had been deleted (DCM-2Tg×IP3-R(2)−/−) and transverse aortic constriction was performed on IP3-R(2)−/− mice. Hearts from DCM-2Tg mice and DCM-2Tg×IP3-R(2)−/− were similar in terms of chamber dilatation, atrial enlargement, and ventricular wall thinning. Electrophysiological changes were also similar in the DCM-2Tg mice, with and without IP3-R(2). Deletion of IP3-R(2) did not alter the progression of heart failure, because DCM-2Tg mice with and without IP3-R(2) had similarly reduced contractility, increased lung congestion, and atrial thrombus, and both strains died between 10 and 12 weeks of age. Loss of IP3-R(2) did not alter the progression of hypertrophy after transverse aortic constriction. Conclusions—We conclude that IP3-R(2) do not contribute to the progression of DCM or pressure overload hypertrophy, despite increased expression and heightened generation of the ligand, Ins(1,4,5)P3.

Postnatal Loss of Kindlin-2 Leads to Progressive Heart Failure

Background—The striated muscle costamere, a multiprotein complex at the boundary between the sarcomere and the sarcolemma, plays an integral role in maintaining striated muscle structure and function. Multiple costamere-associated proteins, such as integrins and integrin-interacting proteins, have been identified and shown to play an increasingly important role in the pathogenesis of human cardiomyopathy. Kindlin-2 is an adaptor protein that binds to the integrin &bgr; cytoplasmic tail to promote integrin activation. Genetic deficiency of Kindlin-2 results in embryonic lethality, and knockdown of the Kindlin-2 homolog in Caenorhabditis elegans and Danio rerio suggests that it has an essential role in integrin function and normal muscle structure and function. The precise role of Kindlin-2 in the mammalian cardiac myocyte remains to be determined. Methods and Results—The current studies were designed to investigate the role of Kindlin-2 in the mammalian heart. We generated a series of cardiac myocyte–specific Kindlin-2 knockout mice with excision of the Kindlin-2 gene in either developing or adult cardiac myocytes. We found that mice lacking Kindlin-2 in the early developing heart are embryonic lethal. We demonstrate that deletion of Kindlin-2 at late gestation or in adult cardiac myocytes resulted in heart failure and premature death, which were associated with enlargement of the heart and extensive fibrosis. In addition, integrin &bgr;1D protein expression was significantly downregulated in the adult heart. Conclusions—Kindlin-2 is required to maintain integrin &bgr;1D protein stability. Postnatal loss of Kindlin-2 from cardiac myocytes leads to progressive heart failure, showing the importance of costameric proteins like Kindlin-2 for homeostasis of normal heart function.

Generation and Characterization of a Mouse Model Harboring the Exon-3 Deletion in the Cardiac Ryanodine Receptor

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del+/−) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del+/− mice are not susceptible to CPVT. Ex3-del+/− cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca2+ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del+/− mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del+/− mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del+/− mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion.

HSPB7 is indispensable for heart development by modulating actin filament assembly

Significance Sarcomeres, the contractile units of striated muscle, are composed of thick and thin/actin filaments. Thin filament length is closely associated with specific contractile properties of individual muscles, and it is tightly controlled by actin binding proteins. However, it is still unclear how these proteins work in concert to maintain proper thin filament length and whether there are additional factors involved. In this study, we found that deleting HSPB7 resulted in uncontrolled elongation of actin filaments and the formation of atypical actin filament bundles in cardiomyocytes. Biochemical studies revealed a previously unsuspected function of HSPB7 in interacting with and limiting actin monomer availability for actin filament polymerization, giving mechanistic insight into the etiology of aberrant sarcomeres observed in HSPB7 null heart. Small heat shock protein HSPB7 is highly expressed in the heart. Several mutations within HSPB7 are associated with dilated cardiomyopathy and heart failure in human patients. However, the precise role of HSPB7 in the heart is still unclear. In this study, we generated global as well as cardiac-specific HSPB7 KO mouse models and found that loss of HSPB7 globally or specifically in cardiomyocytes resulted in embryonic lethality before embryonic day 12.5. Using biochemical and cell culture assays, we identified HSPB7 as an actin filament length regulator that repressed actin polymerization by binding to monomeric actin. Consistent with HSPB7’s inhibitory effects on actin polymerization, HSPB7 KO mice had longer actin/thin filaments and developed abnormal actin filament bundles within sarcomeres that interconnected Z lines and were cross-linked by α-actinin. In addition, loss of HSPB7 resulted in up-regulation of Lmod2 expression and mislocalization of Tmod1. Furthermore, crossing HSPB7 null mice into an Lmod2 null background rescued the elongated thin filament phenotype of HSPB7 KOs, but double KO mice still exhibited formation of abnormal actin bundles and early embryonic lethality. These in vivo findings indicated that abnormal actin bundles, not elongated thin filament length, were the cause of embryonic lethality in HSPB7 KOs. Our findings showed an unsuspected and critical role for a specific small heat shock protein in directly modulating actin thin filament length in cardiac muscle by binding monomeric actin and limiting its availability for polymerization.

aPKCζ-dependent Repression of Yap is Necessary for Functional Restoration of Irradiated Salivary Glands with IGF-1

Xerostomia and salivary hypofunction often result as a consequence of radiation therapy for head and neck cancers, which are diagnosed in roughly 60,000 individuals every year in the U.S. Due to the lack of effective treatments for radiation-induced salivary hypofunction, stem cell-based therapies have been suggested to regenerate the irradiated salivary glands. Pharmacologically, restoration of salivary gland function has been accomplished in mice by administering IGF-1 shortly after radiation treatment, but it is not known if salivary stem and progenitor cells play a role. We show that radiation inactivates aPKCζ and promotes nuclear redistribution of Yap in a population of label-retaining cells in the acinar compartment of the parotid gland (PG)– which comprises a heterogeneous pool of salivary progenitors. Administration of IGF-1 post-radiation maintains activation of aPKCζ and partially rescues Yap’s cellular localization in label retaining cells, while restoring salivary function. Finally, IGF-1 fails to restore saliva production in mice lacking aPKCζ, demonstrating the importance of the kinase as a potential therapeutic target.

No Contribution of IP 3 -R(2) to Disease Phenotype in Models of Dilated Cardiomyopathy or Pressure Overload Hypertrophy

Background—We investigated the contribution of inositol(1,4,5)-trisphosphate (Ins(1,4,5)P3 [IP3]) receptors (IP3-R) to disease progression in mouse models of dilated cardiomyopathy (DCM) and pressure overload hypertrophy. Mice expressing mammalian sterile 20–like kinase and dominant-negative phosphatidylinositol-3-kinase in heart (Mst1×dn-PI3K-2Tg; DCM-2Tg) develop severe DCM and conduction block, associated with increased expression of type 2 IP3-R (IP3-R(2)) and heightened generation of Ins(1,4,5)P3. Similar increases in Ins(1,4,5)P3 and IP3-R(2) are caused by transverse aortic constriction. Methods and Results—To evaluate the contribution of IP3-R(2) to disease progression, the DCM-2Tg mice were further crossed with mice in which the type 2 IP3-R (IP3-R(2)−/−) had been deleted (DCM-2Tg×IP3-R(2)−/−) and transverse aortic constriction was performed on IP3-R(2)−/− mice. Hearts from DCM-2Tg mice and DCM-2Tg×IP3-R(2)−/− were similar in terms of chamber dilatation, atrial enlargement, and ventricular wall thinning. Electrophysiological changes were also similar in the DCM-2Tg mice, with and without IP3-R(2). Deletion of IP3-R(2) did not alter the progression of heart failure, because DCM-2Tg mice with and without IP3-R(2) had similarly reduced contractility, increased lung congestion, and atrial thrombus, and both strains died between 10 and 12 weeks of age. Loss of IP3-R(2) did not alter the progression of hypertrophy after transverse aortic constriction. Conclusions—We conclude that IP3-R(2) do not contribute to the progression of DCM or pressure overload hypertrophy, despite increased expression and heightened generation of the ligand, Ins(1,4,5)P3.

No Contribution of IP3-R(2) to Disease Phenotype in Models of Dilated Cardiomyopathy or Pressure Overload HypertrophyClinical Perspective

Background—We investigated the contribution of inositol(1,4,5)-trisphosphate (Ins(1,4,5)P3 [IP3]) receptors (IP3-R) to disease progression in mouse models of dilated cardiomyopathy (DCM) and pressure overload hypertrophy. Mice expressing mammalian sterile 20–like kinase and dominant-negative phosphatidylinositol-3-kinase in heart (Mst1×dn-PI3K-2Tg; DCM-2Tg) develop severe DCM and conduction block, associated with increased expression of type 2 IP3-R (IP3-R(2)) and heightened generation of Ins(1,4,5)P3. Similar increases in Ins(1,4,5)P3 and IP3-R(2) are caused by transverse aortic constriction. Methods and Results—To evaluate the contribution of IP3-R(2) to disease progression, the DCM-2Tg mice were further crossed with mice in which the type 2 IP3-R (IP3-R(2)−/−) had been deleted (DCM-2Tg×IP3-R(2)−/−) and transverse aortic constriction was performed on IP3-R(2)−/− mice. Hearts from DCM-2Tg mice and DCM-2Tg×IP3-R(2)−/− were similar in terms of chamber dilatation, atrial enlargement, and ventricular wall thinning. Electrophysiological changes were also similar in the DCM-2Tg mice, with and without IP3-R(2). Deletion of IP3-R(2) did not alter the progression of heart failure, because DCM-2Tg mice with and without IP3-R(2) had similarly reduced contractility, increased lung congestion, and atrial thrombus, and both strains died between 10 and 12 weeks of age. Loss of IP3-R(2) did not alter the progression of hypertrophy after transverse aortic constriction. Conclusions—We conclude that IP3-R(2) do not contribute to the progression of DCM or pressure overload hypertrophy, despite increased expression and heightened generation of the ligand, Ins(1,4,5)P3.