Hypertrophic Cardiomyopathy (HCM) has been related to many different mutations in more than 20 different, mostly sarcomeric proteins. While development of the HCM-phenotype is thought to be triggered by the different mutations, a common mechanism remains elusive. Studying missense-mutations in the ventricular beta-myosin heavy chain (β-MyHC, MYH7) we hypothesized that significant contractile heterogeneity exists among individual cardiomyocytes of HCM-patients that results from cell-to-cell variation in relative expression of mutated vs. wildtype β-MyHC. To test this hypothesis, we measured force-calcium-relationships of cardiomyocytes isolated from myocardium of heterozygous HCM-patients with either β-MyHC-mutation Arg723Gly or Arg200Val, and from healthy controls. From the myocardial samples of the HCM-patients we also obtained cryo-sections, and laser-microdissected single cardiomyocytes for quantification of mutated vs. wildtype MYH7-mRNA using a single cell RT-qPCR and restriction digest approach. We characterized gene transcription by visualizing active transcription sites by fluorescence in situ hybridization of intronic and exonic sequences of MYH7-pre-mRNA. For both mutations, cardiomyocytes showed large cell-to-cell variation in Ca++-sensitivity. Interestingly, some cardiomyocytes were essentially indistinguishable from controls what might indicate that they had no mutant β-MyHC while others had highly reduced Ca++-sensitivity suggesting substantial fractions of mutant β-MyHC. Single-cell MYH7-mRNA-quantification in cardiomyocytes of the same patients revealed high cell-to-cell variability of mutated vs. wildtype mRNA, ranging from essentially pure mutant to essentially pure wildtype MYH7-mRNA. We found 27% of nuclei without active transcription sites which is inconsistent with continuous gene transcription but suggests burst-like transcription of MYH7. Model simulations indicated that burst-like, stochastic on/off-switching of MYH7 transcription, which is independent for mutant and wildtype alleles, could generate the observed cell-to-cell variation in the fraction of mutant vs. wildtype MYH7-mRNA, a similar variation in β-MyHC-protein, and highly heterogeneous Ca++-sensitivity of individual cardiomyocytes. In the long run, such contractile imbalance in the myocardium may well induce progressive structural distortions like cellular and myofibrillar disarray and interstitial fibrosis, as they are typically observed in HCM.
Secretory Pathway Ca 2þ -ATPases (SPCA) are P-type pumps that localize to the Golgi and post-Golgi vesicular compartments into which they sequester Ca 2þ and Mn 2þ ions required for protein processing, glycosylation and sorting. There are two isoforms in human that play distinct roles in breast cancer, a leading cause of death in women worldwide. Previously, we showed that SPCA2, but not SPCA1, interacts with the Ca 2þ channel Orai1 to initiate storeindependent Ca 2þ entry, which promotes tumorigenesis. We sought to understand the roles of SPCA1 and SPCA2 in breast cancer and how they correlate with the different subtypes and with epithelial-mesenchymal transition (EMT). SPCA2 expression is greatly amplified in luminal subtypes but has a lower expression in basal subtypes, with opposite expression patterns for SPCA1. We present analysis of patient survival and microarray data for both SPCA1 and SPCA2 expression, and evaluate isoform-specific roles in two different breast cancer cell lines, which represent epithelial (MCF7) and post-EMT (MDA-MB-231) subtypes. Using knockdown and overexpression approaches, we correlate isoform expression with cell proliferation, tumorsphere formation, and plasma membrane trafficking of a cell adhesion protein critical for tumorigenesis, E-cadherin. From these studies, we conclude that SPCA2 promotes oncogenesis by regulating E-cadherin and that SPCA2 down regulation may be critical for metastasis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.