Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities
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This manuscript will be of interest to scientists who study cardiomyocyte homeostasis and contraction. It assesses the functional consequences of cardiomyocyte-specific knockout of Palladin, leading to the identification of a compensation mechanism when Palladin is deleted in embryogenesis, but not in adulthood. In addition, the authors identified new Palladin interactors, revealing a role for Palladin in the maintenance of intercalated disc structure.
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Abstract
Palladin (PALLD) belongs to the PALLD/myopalladin (MYPN)/myotilin family of actin-associated immunoglobulin-containing proteins in the sarcomeric Z-line. PALLD is ubiquitously expressed in several isoforms, and its longest 200 kDa isoform, predominantly expressed in striated muscle, shows high structural homology to MYPN. MYPN gene mutations are associated with human cardiomyopathies, whereas the role of PALLD in the heart has remained unknown, partly due to embryonic lethality of PALLD knockout mice. In a yeast two-hybrid screening, CARP/Ankrd1 and FHOD1 were identified as novel interaction partners of PALLD’s N-terminal region. To study the role of PALLD in the heart, we generated conditional (cPKO) and inducible (cPKOi) cardiomyocyte-specific PALLD knockout mice. While cPKO mice exhibited no pathological phenotype, ablation of PALLD in adult cPKOi mice caused progressive cardiac dilation and systolic dysfunction, associated with reduced cardiomyocyte contractility, intercalated disc abnormalities, and fibrosis, demonstrating that PALLD is essential for normal cardiac function. Double cPKO and MYPN knockout (MKO) mice exhibited a similar phenotype as MKO mice, suggesting that MYPN does not compensate for the loss of PALLD in cPKO mice. Altered transcript levels of MYPN and PALLD isoforms were found in myocardial tissue from human dilated and ischemic cardiomyopathy patients, whereas their protein expression levels were unaltered.
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Author Response:
Reviewer #1 (Public Review):
In this manuscript, Mastrototaro et al. perform a series of experiments in transgenic murine models assessing the function of Palladin (PALLD) in the heart. Global PALLD KOs are embryonic lethal, precluding the assessment of the roles of this protein in adulthood. To circumvent this limitation, the authors generated a floxed Palld allele and ablated it with two cardiomyocyte-specific Cres: the constitutively active Myh6-Cre and the tamoxifen-inducible aMHC-MerCreMer. Interestingly, ablation with the constitutive Cre (cKO) did not produce any overt phenotype, but ablation in adulthood (cKOi) resulted in compromised cardiac function. These observations suggest a compensation mechanism that takes place when cardiomyocytes develop in the complete absence of this protein but not when …
Author Response:
Reviewer #1 (Public Review):
In this manuscript, Mastrototaro et al. perform a series of experiments in transgenic murine models assessing the function of Palladin (PALLD) in the heart. Global PALLD KOs are embryonic lethal, precluding the assessment of the roles of this protein in adulthood. To circumvent this limitation, the authors generated a floxed Palld allele and ablated it with two cardiomyocyte-specific Cres: the constitutively active Myh6-Cre and the tamoxifen-inducible aMHC-MerCreMer. Interestingly, ablation with the constitutive Cre (cKO) did not produce any overt phenotype, but ablation in adulthood (cKOi) resulted in compromised cardiac function. These observations suggest a compensation mechanism that takes place when cardiomyocytes develop in the complete absence of this protein but not when cardiomyocytes develop in a wild-type background and are deprived of this protein after achieving full maturation. These experiments were complemented with yeast two-hybrid techniques to identify novel partners that bind to a region of PALLD for each no interactants had been previously identified. Experiments in human samples revealed an upregulation of PALLD transcripts in the hearts of patients.
This manuscript adds important information to our understanding of sarcomeric proteins. Data are generally of good quality and well presented in figures. The numbers of animals in echocardiographic studies are also adequate for proper conclusions. Authors achieve most of their goals, including the identification of novel partners of PALLD and the identification of a requirement for PALLD in cardiomyocytes for normal heart function. However, given that all experiments performed in this study were focused on the loss-of-function of PALLD, it is not clear what is the relevance of the PALLD upregulation observed in human patients. Authors should clearly state this limitation in their results.
Considering that authors have observed evidence for nuclear PALLD, which could hint at potential major gene expression changes when this protein is ablated, it would be interesting to perform an unbiased assessment of transcriptional alterations (RNA-seq) in cardiomyocytes isolated from control and cKOi hearts. In addition, to test if the compensation observed in the embryonic cKO involves mechanisms of transcriptional adaptation, it would be interesting to compare RNA-seq results from cKOi and cKO (genes encoding proteins similar to PALLD that are upregulated in cKO but not cKOi cardiomyocytes would be very strong candidates). However, these transcriptomic data are not essential to support current findings and can be performed in follow-up studies.
We agree with the reviewer that it would be interesting to perform RNA-Seq on isolated cardiomyocytes from cPKOi mice and we are in fact planning to do this in a follow-up study.
Reviewer #2 (Public Review):
The role of the actin-binding protein palladin (PALLD) in cardiomyocyte development, growth, and function has not been defined. In order to address this question, the authors first identified that CARP and FHOD1 interact with PALLD in cardiomyocytes. They then performed cardiomyocyte selective deletion of PALLD in embryonic and adult mice and discovered that deletion of PALLD in adult mice leads to dilated cardiomyopathy (DCM) and intercalated disc ultrastructural changes. In contrast, embryonic deletion of cardiomyocyte PALLD did not cause a cardiomyopathy phenotype in neonatal or adult animals.
1. The divergent cardiac phenotypes of the embryonic deletion of cardiomyocyte PALLD (no cardiomyopathy) versus the adult deletion of cardiomyocyte PALLD (dilated cardiomyopathy(DCM)) is an interesting result. The authors speculate that embryonic deletion of PALLD induces compensatory pathways that prevent the development of adult cardiomyopathy in these mice. However, these compensatory pathways remain unexplored.
2. The authors discovered that mice with adult cardiomyocyte deletion of PALLD had significant changes in the cardiomyocyte intercalated disc (ICD) ultrastructure. They suggest these changes in ICD ultrastructure contribute to DCM formation in the adult PALLD deletion mice (line 270). However, it remains unclear if these changes in ICD ultrastructure are specific to mice with adult deletion of PALLD.
3. The different transgenic Cre mouse lines may be an alternative explanation for the divergent cardiac phenotypes in the embryonic versus adult deletion of cardiomyocyte PALLD. The tamoxifen dose administered for the inducible Myh6:MerCreMer mice was 30mg/kg/day x 5 which has been reported to lead to the induction of cardiomyocyte DNA damage response pathways (Dis Model Mech. 2013 Nov; 6(6): 1459-1469, J Cardiovasc Aging 2022;2:8). The electron micrograph experiments in Figure 5 did not include a group of Myh6:MerCreMer mice administered tamoxifen. The authors only compared PALLD fl/fl and Myh6:MerCreMer/PALLD fl/fl mice.In the papers that the Reviewer refers to it was shown that administration of tamoxifen to Myh6:MerCreMer mice at a dose of 30 mg/kg/day for 3 (Bersell et al., Dis Model Mech. 6, 1459-1469, 2013) or 5 days (Rouhi et al., J Cardiovasc Aging 2, 8, 2022) is not associated with apoptosis. Bersell et al., found that amounts ≥40 mg/kg/day for 3 days is associated with apoptosis, and Rouhi et al., showed that injection of 30 mg/kg/day for 5 days causes transient minor changes in gene expression with no discernible effects on cardiac function, myocardial fibrosis, apoptosis, or induction of double-stranded DNA breaks. The reason that we chose to inject tamoxifen at an amount of 30 mg/kg/day for 5 days was in fact that this amount has been shown not to be associated with severe effects and has been widely used in the literature.
4. The apoptosis assessment was performed 24 weeks after administration of tamoxifen to the Myh6:MerCreMer/PALLD fl/fl mice. However, cardiomyocyte apoptosis may have occurred much earlier if it was secondary to Myh6:MerCreMer tamoxifen-induced cardiotoxicity (or related to PALLD deletion).
5. The animal studies in Fig 3D show a DCM phenotype in mice with adult deletion of cardiomyocyte 200kDa PALLD which suggests a potential loss of function mechanism for DCM formation. However, the authors then report in Fig 6 that human DCM heart tissue samples have a ~2.5fold increase in mRNA expression of the 200kDa PALLD transcript which would suggest a possible gain of function mechanism for DCM formation. How do the authors reconcile these divergent results with regard to palladin's role in cardiomyocyte homeostasis and cardiomyopathy formation?In the revised manuscript we demonstrate that the transcriptional changes in PALLD expression are not reflected at the protein level.
Reviewer #3 (Public Review):
This study shows for the first time changes in palladin expression under disease conditions and mRNA alterations in human samples. The authors have identified novel binding partners for the protein as a first step toward determining how palladin mediates its effects in the heart. Finally, through the use of mouse models to decrease palladin expression they identify a crucial role for palladin in the cardiac response to pathological stress, with some interesting findings that show the effects of palladin depend on when the protein is altered.
We appreciate that the Reviewer finds our study interesting. However, we did not show a role of PALLD in the cardiac response to pathological stress. On the contrary, we demonstrated that mice with constitutive knockout of PALLD in the heart (cPKO mice) show no pathological cardiac phenotype either under basal conditions or in response to mechanical pressure overload by transaortic constriction. On the other hand, deletion of PALLD in adult mice resulted in DCM under basal conditions within 8 weeks after tamoxifen induction.
The novel findings of the study are supported by the data presented, but there are several instances where clarification is needed of the conclusions drawn from the data reach beyond what is presented in the Results section.
The focus on only male mice is a significant limitation of the paper, as it is well known that there are profound sex differences in the response to pathological stressors. While the ability to obtain sufficient heart samples from male and female patients may be a reasonable justification for focusing on males, the preclinical mouse model should have been examined in both sexes and the limitation of this choice should be clearly noted in the paper.
Due to the three Rs and the high costs associated with the breeding of the high amount mice required for the project, we chose to focus only on male mice.
In line 537-539, we stated. “All experiments were performed on male mice as females often develop a less severe cardiac phenotype due to the cardioprotective role of estrogen (Brower, Gardner, & Janicki, 2003; Du, 2004).
The changes in myopalladin expression were not measured in the disease model (TAC), which limits the ability to determine if myopalladin was altered in the disease state. This addition would strengthen the study.
We have previously demonstrated that myopalladin protein levels are significantly reduced after TAC in wildtype mice (Figure 6K, L in Filomena et al., eLife 10:e58313, 2021). We did not measure myopalladin levels in cPKO subjected to TAC and unfortunately don’t have tissue from cPKO mice to perform the measurements.
Finally, the myofilament data are presented as evidence that changes in the contractile apparatus are contributors to the observed contractile dysfunction at the organ level. But these studies were conducted using levels of calcium that far exceed what is seen in vivo and, therefore, do not support the conclusion drawn.
The reviewer is right that the myofibril experiments were conducted at Ca2+ concentrations that cannot be reached under the physiological conditions of cardiac contraction. However, the result clearly demonstrates that the intrinsic force generating capacity of the cardiac sarcomeres of cPKOi mice is impaired 8 weeks after TAM independently from any changes in myofilament Ca2+ sensitivity and cardiomyocyte Ca2+ handling. Experiments at lower (more physiological) Ca2+ concentrations would have produced less clear results in the absence of a full investigation of the relation between force and [Ca2+]. Since data demonstrate that cross bridge mechanics and kinetics are not affected, the reported finding supports the idea that a myofibril structural defect is responsible for the lower maximal force of the KO sarcomeres.
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eLife assessment
This manuscript will be of interest to scientists who study cardiomyocyte homeostasis and contraction. It assesses the functional consequences of cardiomyocyte-specific knockout of Palladin, leading to the identification of a compensation mechanism when Palladin is deleted in embryogenesis, but not in adulthood. In addition, the authors identified new Palladin interactors, revealing a role for Palladin in the maintenance of intercalated disc structure.
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Reviewer #1 (Public Review):
In this manuscript, Mastrototaro et al. perform a series of experiments in transgenic murine models assessing the function of Palladin (PALLD) in the heart. Global PALLD KOs are embryonic lethal, precluding the assessment of the roles of this protein in adulthood. To circumvent this limitation, the authors generated a floxed Palld allele and ablated it with two cardiomyocyte-specific Cres: the constitutively active Myh6-Cre and the tamoxifen-inducible aMHC-MerCreMer. Interestingly, ablation with the constitutive Cre (cKO) did not produce any overt phenotype, but ablation in adulthood (cKOi) resulted in compromised cardiac function. These observations suggest a compensation mechanism that takes place when cardiomyocytes develop in the complete absence of this protein but not when cardiomyocytes develop in a …
Reviewer #1 (Public Review):
In this manuscript, Mastrototaro et al. perform a series of experiments in transgenic murine models assessing the function of Palladin (PALLD) in the heart. Global PALLD KOs are embryonic lethal, precluding the assessment of the roles of this protein in adulthood. To circumvent this limitation, the authors generated a floxed Palld allele and ablated it with two cardiomyocyte-specific Cres: the constitutively active Myh6-Cre and the tamoxifen-inducible aMHC-MerCreMer. Interestingly, ablation with the constitutive Cre (cKO) did not produce any overt phenotype, but ablation in adulthood (cKOi) resulted in compromised cardiac function. These observations suggest a compensation mechanism that takes place when cardiomyocytes develop in the complete absence of this protein but not when cardiomyocytes develop in a wild-type background and are deprived of this protein after achieving full maturation. These experiments were complemented with yeast two-hybrid techniques to identify novel partners that bind to a region of PALLD for each no interactants had been previously identified. Experiments in human samples revealed an upregulation of PALLD transcripts in the hearts of patients.
This manuscript adds important information to our understanding of sarcomeric proteins. Data are generally of good quality and well presented in figures. The numbers of animals in echocardiographic studies are also adequate for proper conclusions. Authors achieve most of their goals, including the identification of novel partners of PALLD and the identification of a requirement for PALLD in cardiomyocytes for normal heart function. However, given that all experiments performed in this study were focused on the loss-of-function of PALLD, it is not clear what is the relevance of the PALLD upregulation observed in human patients. Authors should clearly state this limitation in their results.
Considering that authors have observed evidence for nuclear PALLD, which could hint at potential major gene expression changes when this protein is ablated, it would be interesting to perform an unbiased assessment of transcriptional alterations (RNA-seq) in cardiomyocytes isolated from control and cKOi hearts. In addition, to test if the compensation observed in the embryonic cKO involves mechanisms of transcriptional adaptation, it would be interesting to compare RNA-seq results from cKOi and cKO (genes encoding proteins similar to PALLD that are upregulated in cKO but not cKOi cardiomyocytes would be very strong candidates). However, these transcriptomic data are not essential to support current findings and can be performed in follow-up studies.
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Reviewer #2 (Public Review):
The role of the actin-binding protein palladin (PALLD) in cardiomyocyte development, growth, and function has not been defined. In order to address this question, the authors first identified that CARP and FHOD1 interact with PALLD in cardiomyocytes. They then performed cardiomyocyte selective deletion of PALLD in embryonic and adult mice and discovered that deletion of PALLD in adult mice leads to dilated cardiomyopathy (DCM) and intercalated disc ultrastructural changes. In contrast, embryonic deletion of cardiomyocyte PALLD did not cause a cardiomyopathy phenotype in neonatal or adult animals.
1. The divergent cardiac phenotypes of the embryonic deletion of cardiomyocyte PALLD (no cardiomyopathy) versus the adult deletion of cardiomyocyte PALLD (dilated cardiomyopathy(DCM)) is an interesting result. The …
Reviewer #2 (Public Review):
The role of the actin-binding protein palladin (PALLD) in cardiomyocyte development, growth, and function has not been defined. In order to address this question, the authors first identified that CARP and FHOD1 interact with PALLD in cardiomyocytes. They then performed cardiomyocyte selective deletion of PALLD in embryonic and adult mice and discovered that deletion of PALLD in adult mice leads to dilated cardiomyopathy (DCM) and intercalated disc ultrastructural changes. In contrast, embryonic deletion of cardiomyocyte PALLD did not cause a cardiomyopathy phenotype in neonatal or adult animals.
1. The divergent cardiac phenotypes of the embryonic deletion of cardiomyocyte PALLD (no cardiomyopathy) versus the adult deletion of cardiomyocyte PALLD (dilated cardiomyopathy(DCM)) is an interesting result. The authors speculate that embryonic deletion of PALLD induces compensatory pathways that prevent the development of adult cardiomyopathy in these mice. However, these compensatory pathways remain unexplored.
2. The authors discovered that mice with adult cardiomyocyte deletion of PALLD had significant changes in the cardiomyocyte intercalated disc (ICD) ultrastructure. They suggest these changes in ICD ultrastructure contribute to DCM formation in the adult PALLD deletion mice (line 270). However, it remains unclear if these changes in ICD ultrastructure are specific to mice with adult deletion of PALLD.
3. The different transgenic Cre mouse lines may be an alternative explanation for the divergent cardiac phenotypes in the embryonic versus adult deletion of cardiomyocyte PALLD. The tamoxifen dose administered for the inducible Myh6:MerCreMer mice was 30mg/kg/day x 5 which has been reported to lead to the induction of cardiomyocyte DNA damage response pathways (Dis Model Mech. 2013 Nov; 6(6): 1459-1469, J Cardiovasc Aging 2022;2:8). The electron micrograph experiments in Figure 5 did not include a group of Myh6:MerCreMer mice administered tamoxifen. The authors only compared PALLD fl/fl and Myh6:MerCreMer/PALLD fl/fl mice.
4. The apoptosis assessment was performed 24 weeks after administration of tamoxifen to the Myh6:MerCreMer/PALLD fl/fl mice. However, cardiomyocyte apoptosis may have occurred much earlier if it was secondary to Myh6:MerCreMer tamoxifen-induced cardiotoxicity (or related to PALLD deletion).
5. The animal studies in Fig 3D show a DCM phenotype in mice with adult deletion of cardiomyocyte 200kDa PALLD which suggests a potential loss of function mechanism for DCM formation. However, the authors then report in Fig 6 that human DCM heart tissue samples have a ~2.5fold increase in mRNA expression of the 200kDa PALLD transcript which would suggest a possible gain of function mechanism for DCM formation. How do the authors reconcile these divergent results with regard to palladin's role in cardiomyocyte homeostasis and cardiomyopathy formation? -
Reviewer #3 (Public Review):
This study shows for the first time changes in palladin expression under disease conditions and mRNA alterations in human samples. The authors have identified novel binding partners for the protein as a first step toward determining how palladin mediates its effects in the heart. Finally, through the use of mouse models to decrease palladin expression they identify a crucial role for palladin in the cardiac response to pathological stress, with some interesting findings that show the effects of palladin depend on when the protein is altered.
The novel findings of the study are supported by the data presented, but there are several instances where clarification is needed of the conclusions drawn from the data reach beyond what is presented in the Results section.
The focus on only male mice is a significant …
Reviewer #3 (Public Review):
This study shows for the first time changes in palladin expression under disease conditions and mRNA alterations in human samples. The authors have identified novel binding partners for the protein as a first step toward determining how palladin mediates its effects in the heart. Finally, through the use of mouse models to decrease palladin expression they identify a crucial role for palladin in the cardiac response to pathological stress, with some interesting findings that show the effects of palladin depend on when the protein is altered.
The novel findings of the study are supported by the data presented, but there are several instances where clarification is needed of the conclusions drawn from the data reach beyond what is presented in the Results section.
The focus on only male mice is a significant limitation of the paper, as it is well known that there are profound sex differences in the response to pathological stressors. While the ability to obtain sufficient heart samples from male and female patients may be a reasonable justification for focusing on males, the preclinical mouse model should have been examined in both sexes and the limitation of this choice should be clearly noted in the paper.
The changes in myopalladin expression were not measured in the disease model (TAC), which limits the ability to determine if myopalladin was altered in the disease state. This addition would strengthen the study.
Finally, the myofilament data are presented as evidence that changes in the contractile apparatus are contributors to the observed contractile dysfunction at the organ level. But these studies were conducted using levels of calcium that far exceed what is seen in vivo and, therefore, do not support the conclusion drawn.
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