Dilated Cardiomyopathy: the Mechanisms and Genes Behind the Disease Dilated

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 Cardiomyopathy (DCM) is a disorder that can cause sudden death, as well as heart failure. 1 out of 2500, are affected by DCM (Zhao et al 1). It is centralized to the left ventricle in most cases and symptoms of this disease produce an enlargement of the left ventricle and normal heart functions are restricted such as contractility. One important factor that is involved in the contraction of heart muscles is a complex called Cardiac Troponin. This is a specialized troponin complex and within this complex are different subunits. Within these different subunits, each has a specific role that contributes to the contraction process of the heart (Duke et al 2). In the past years, research has expanded and more gene mutations and variants have been identified, especially within this complex in particular. In the review below, DCM will introduce a variety of gene mutations that have been discovered to support the causation of this disease, and a particular subunit, Troponin T, within the protein complex transcribed by TNNT 2 gene, has a wide variety of variant mutations throughout three articles. Also, the importance of future research includes genetic screening and the impacts this could have in the field of Cardiomyopathy.

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Dilated Cardiomyopathy (DCM) is a genetic condition that affects the hearts of patients, and more specifically the left ventricle. Patients with this condition see an enlargement of this area of the heart and effects in their systolic measurements, which primarily focuses on the amount of contraction seen in the muscle. The pathogenesis of this disease is genetic and is inherited by Mendelian Autosomal Dominant Patterns, which means that at least one parent is affected and this is passed on to the offspring. There are many genes thought to be candidates for the causation of this disease, but mutations in more than 50 genes have been documented thus far. In the cohort study, researchers focused on the Yumman population in China. 7 novel mutations were identified and researchers identified three variants that produced effects in the experiment, as well as a confirmation of two known mutations of this disease (Zhao et al 1). Those with DCM are most at risk for heart failure and sudden cardiac death. In this study, researchers aimed at identifying genes that were most likely the root of causations, and implemented that necessity for genetic testing as a useful tool in diagnosis or predicting future diagnosis for family members.

Methods & Materials

Researchers first began their study by gathering a pool of 21 participants from the southern and western regions of Yuman, China, as these individuals were diagnosed with DCM. These patients were included as they met specific guidelines. They underwent further clinical testing to help researchers identify the phenotypes of the patients. These phenotypes focused looked at Left Ventricle Ejection Fraction (LVEF), which is the percentage of blood that flows through after a contraction, as well as the diameter of the Left Ventricle (LVED). Next, to focus on specific genes, researchers took peripheral blood lymphocytes from the patients and extracted genomic DNA to isolate the amplicons. From this set of data, 25 genes were selected as candidate genes and amplicons to move forward in the experiment. From these candidate genes, variants in these gene mutations were focused on to see which were synonymous, nonsynonymous, and those with uncertain significance. They did this by comparing to several national databases such as the 1000 Genomes Project, Human Gene Mutation Database, and the National Heart, Lung, and Blood Institute Exome Sequencing Project. Those mutations that showed several alterations or effects on functions were then analyzed by a variety of tools, and further testing was done as a reconfirmation for the gene mutations selected through the use of traditional Sanger Sequencing (Zhao et al 2).


Of the 21 patients (15 male, 6 female) all showed an enlargement of the left ventricle and the LVESD. Of the patients used in the study, 57.2% of patients mutations were found, and when looking at patients carrying mutation variants, 66.7% expressed this high frequency in sarcomeric genes (Zhao et al 6). Of those genes that were sequenced and mapped, all proved to be successful as 243 variant mutations were identified and 12 average variations were found per patient. After a comparison of variants found in this study and the literature of the databases 12 possible pathogenic mutations were found and were later confirmed by the Sanger sequence experiment (Zhao et al 3). Of the 12 identified pathogenic gene mutations, 7 novel mutations were found that were not included in the national databases. The highest frequencies were seen in sarcomeric genes at 66.7%, and the second-highest frequency seen in this study focused on the cytoskeleton genes at 25% (Zhao et al 6).


Researchers used clinical evaluations to reconfirm phenotypes of patients with DCM and further went on to exploit what pathogenic gene mutations could be the driving force behind this disorder. They discover 7 novel genes that they considered note-worthy as that had not previously been identified before this experiment. The gene mutations ranged from those that affect sarcomeric genes such as Tnnt-2, and those that affected the cytoskeleton such as (MYNP). These genes had severe alterations in amino sequences that would then affect the transcription and translation of proteins critical for contractions of the heart muscles (Zhao et al 6). Changes in sequence would vary in severity that would affect the function of the proteins. Researchers aimed this study to approach which genes were the possible driving forces behind this disorder and used genetic sequencing as a fundamental tool to support their claims. They argue that personalized genetic sequencing would be a vital asset in determining next-steps for possible prevention methods. However, although it may be crucial, they understand the cost and time inefficiency of this process. Within the study, they began with limited data that would benefit the experiment as it would be very expensive to analyze all data available, so modifications had to be made, and then sequence genes that had secure footing were processed through and then resequenced as a confirmation. They suggested future research be spent on making genetic sequencing more time and cost-efficient as this could be crucial to a patient in need, as well as growing background knowledge for this disease.

Gene-Targeted Mice with the Human Troponin T R141W Mutation Develop Dilated Cardiomyopathy with Calcium Desensitization.


As previously discussed, DCM has many possible genes that can cause the disorder to develop. Those with this disorder have a limited rate of survival after diagnosis with 25% at one year, and 50% after 5 years (Ramratnam et al 2). However, most studies have been performed in vitro to demonstrate the mechanism behind the mutations with progress slowly being made. One sarcomeric gene, in particular, has been the target of several studies because calcium desensitization at irregular rates has been directed as the cause of a mutation. A variant mutation in the TNNT2 gene, (R141W) mutation, and the focus of this study. Researchers have developed model systems that were heterozygous for this allele type (R141W/+). The focus of the study was to produce these mice positive for this allele type, and then see the effects of gene dosage on phenotype, differences of this disease on genders, the molecular mechanism behind the remodeling of the heart, and lastly, the effects of Calcium Homeostasis.

Methods & Materials

To begin the experiment, researchers had to introduce the mutation into the experimental animal and had done so homologous recombination in derived ES cells and was generated in Exon 9 by PCR. Once a series of steps to incorporate this mutation into ES cells, and target cells were isolated and these cells contained one mutant allele. These cells were placed in mice that were then bred together with 129/SvEv strain EIIa-Cre recombinase transgenic mice to produce offspring that would express the desired genotype (R141W/+). These mice were then bred again once reverse sequencing confirmed the desired genotype was obtained, to produce mice that were homozygous for the genotype. For these genotypes, specific phenotypes such as an enlargement of the heart was monitored, as well as echocardiograms were performed These were run at 6, 12, 30 weeks. Immunoblotting was performed as well to test to see if specific proteins were present or not for pathways involved in heart failure. After these tests were performed, calcium interaction mechanisms were tested by placing strips of muscles from the hearts of the mice in different concentrations of free-floating calcium ions and an activation solution to test the amount of normalized force produced as well as tests were performed to test action potentials of the euthanized mice (Ramratnam et al 3-6).


In gene-targeted mice with the mutation, reverse sequencing was performed and show two peaks at the CGC and TGG codons, which respectively represented the wildtype Arg residue and the mutate Trp mutation (Ramratnam et al 6). Once mice with the correct mutation were confirmed, researchers identified significant increases in the left ventricle systolic diameter of the mice hearts as well as a decrease in the relationship between the amount of pumping of the heart before and after contraction, which they refer to fractional shortening (FS) in the study. Those wildtypes for the mutation had 37∓3, 35∓2, and 35∓2 percentages for 6,12 and 30 weeks respectively. Those heterozygous for the mutation showed 23∓2, 25∓1, and 22 ∓1 percentages for 6, 12 and 30 weeks respectively (Ramratnam et al 8). When focusing on molecular mechanisms by the immunoblot analysis, researchers obtained data that suggested Calcineurin, a Ca2+ regulated phototase associated with heart failure, was elevated significantly compared to normal wild type (3.1∓.12). Results also showed upregulation and downregulation in particular pathways associated with heart failure (Ramratnam et al 9).


In conclusion, the mice with the mutant genotype expressed human characteristics seen in DCM in an animal model. They had seen phenotypes develope that this genotype is responsible for producing. Such as drastic differences in percentages during the duration of the study between the control and experimental groups. However, researchers mentioned particular data points mentioned with different expression levels may produce a range of phenotype expressions. This would confirm that the mice produced the desired mutant gene expression which supports TNNT-2 and the variant mutation type R141W is driver for DCM. Also, signalling pathways were affected as protein expression were increased such as calcineurin, which will lead to calcium desensitization and eventually lead to hypertrophy, or the enlargement of an organ. In this case, it is the LV of the heart. Overall, researchers’ data supported that R141W variant mutations contribute to DCM, and found variables between male and female, as well as the amount of gene expression can contribute to variable severities. They suggest other studies move forward and focus on the different pathways affected by these mutations to expand research on the molecular mechanisms of cardiomyopathy.

Cardiomyopathy-Causing Deletion K210 in Cardiac Troponin T Alters Phosphorylation Propensity of Sarcomeric Proteins


A special complex, Cardiac Troponin, is a sensor for calcium that is involved in myofilaments in muscle contraction of the heart. It is composed of different subunits, and one in particular troponin-T is responsible for binding to tropomyosin on the muscle fiber to act as an anchor and prevent myosin from binding to actin during the contraction process. However, normal function is stopped when mutations occur, and genetic studies have found over 37 mutations within this specific subunit of the complex. Mutations can cause different forms of cardiomyopathy, however, this study focused in on DCM and the deletion of lysine 210 (Duke et al 2 ) Researchers predict that molecular mechanisms, such as phosphorylation of cardiac sarcomeric proteins will be a determining factor for the disease when compared to a wild type model for this experiment.

Methods & Materials

Researchers produced the desired mutation of this study, and did purification sequences on troponin complexes (cTn) and focused on troponin T (cTnT). With this purified product, kinases assays were performed at Thr203 ND Thr284 sites with different concentrations of cTnT in wildtype (WT), heterozygous (HT) and homozygous mice (HM) hearts for the mutation. Another test was performed on freshly isolated myofibers of WT,HT, HM to observe the phosphorylation trends over an 8-24 week period at specific sites on the protein. Also, these sites were tested again with phospho-specific antibodies to check for residues left over. Researchers also developed a 3-D model of a wild type protein and a mutated protein through a program and then ran statistical analyses (Duke et al 2-3).


In the kinase assays, Thr203 regions were affected differently in phosphorylation numbers of concentration and time duration when alone (cTnT) and when compared with the complex as a whole (cTn-WT). In each phosphorylation vs time graph displayed, there was a significant decrease in ability by cTnT-WT alone in the process of phosphorylation, but when cTn-k210 was measured, there was a significant increase in levels than previously seen at respective protein sites. Phosphorylation patterns in the isolated myofibers increased at Thr203 and Thr 284 sites, and decreased at other sites on the protein affecting these sites as well. When producing the 3-D models of wild type and mutant type, the configuration and structure were noticeable different, which would in turn affect the over function efficiency of the protein (Duke et al 12-18).


As differences in substrate concentrations of the subunit cTnT and the complex as a whole appeared, this would affect the enhancing of the PKC pathway, which would affect calcium levels and therefore, this would also affect the contractile properties as well. When analyzing 3-D models, there was a direct correlation to protein function. Researchers noticed that an increase in phosphorylation of PKC in the region of Thr203 due to the deletion of lysine support that binding cTnT to Thr203 will increase calcium desensitization and this will limit contraction properties in sarcomeres. The effects of this also supported affecting nearby regions (Duke et al 7-8). Researchers admit that mutations in the cTnT subunit of the complex may not be responsible for the entirety of the disorder, but plays a large role in contribution. Overall, this study had evidence to support the deletion of lysine would negatively impact the contraction properties that correlate with DCM and overall normal function which would develop DCM symptoms. They were able to showcase in one of the first studies that nearby regions can be affected as well on a protein showcased with Thr203 experiments. Similar to the results, most genes affected, either in the pathways of the regions of the protein, were involved to be sarcomeric genes. Future research that want to be achieved is to find a generalized gene that causes cardiomyopathy across the board, so in a case such as these, treatments that would be developed could be started immediately rather than later.

Overall Conclusion

Overall, each paper referenced DCM and the impact that this disease may cause in patients. Each study referenced as well that there was an enlargement of the LV as well as heart failure. There are a variety of factors that contribute to this disorder such as genes associated with sarcomeres, cytoskeletal regions in the heart, nuclear proteins and so on. Mutations within the TNNT-2 gene have many variants and two were discussed previously, R141W and Lysine 210. Challenges that may be faced by the audience when understanding these papers, is interpreting the graphs and analyzing them as this was difficult for me. Secondly, the audience should have a general background in biology and genetics as terms and phrases used are that area of study dense. Future testing mentioned focused on the importance of genetic screening so early symptoms could be addressed and improve the efficiency of treatments, as well as in general to build an understanding behind the mechanisms of DCM. If further research is conducted, then possible treatments can be developed in response to this disease.

Works Cited

  1. Duke, Liliana S., et al. “Cardiomyopathy-Causing Deletion K210 in Cardiac Troponin T Alters
  2. Phosphorylation Propensity of Sarcomeric Proteins.” Biophysical Journal, vol. 98, no. 3, May 2010, doi:10.1016/j.bpj.2009.12.1906.
  3. Ramratnam, Mohun, et al. “Gene-Targeted Mice with the Human Troponin T R141W Mutation
  4. Develop Dilated Cardiomyopathy with Calcium Desensitization.” Plos One, vol. 11, no.
  5. 12, 9 July 2016, pp. 1–23., doi:10.1371/journal.pone.0167681.
  6. Zhao, Yue, et al. “Targeted next-Generation Sequencing of Candidate Genes Reveals Novel
  7. Mutations in Patients with Dilated Cardiomyopathy.” International Journal of Molecular
  8. Medicine, vol. 36, no. 6, 7 Sept. 2015, pp. 1479–1486., doi:10.3892/ijmm.2015.2361.

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