Infantile fiber-type I hypotrophy with simultaneously occurring severe onset of cardiomyopathy were previously reported in Dutch and Italian families and genetically linked to the MYL2 gene encoding for the human myosin regulatory light chain MLC2ventr/slow expressed in the ventricles and in slow-twitch skeletal muscles. Shortly after birth the patients experienced progressive slow-twitch skeletal myopathy and ultimately died of heart failure between 4 and 6 months of age. Dominant mutations in MYL2 have been known to cause familial hypertrophic cardiomyopathy (FHC) of extensive diversity in the course of the disease, age of onset and severity of symptoms. The mutation-specific dysregulation of the molecular events that trigger pathological remodeling of the heart, will be assessed using our transgenic (Tg) mice expressing the malignant: R58Q and D166V and benign: K104E mutations in MLC2ventr/slow. In addition to cardiac phenotypes, this application for the first time will include the slow-twitch skeletal muscle and the study of the splice site IVS6-1 mutation in MYL2 shown to cause severe myopathy in humans and premature death of IVS6-1-homozygous patients.
AIM 1 : Identify molecular mechanisms responsible for cardioskeletal dysfunction caused by MLC2ventr/slow mutations. We hypothesize that the mutation-induced structural changes trigger pathological remodeling of the heart and slow skeletal muscle leading to altered contractility and cardioskeletal myopathy. Proteomics study will be employed to identify the signaling pathways involved in cardioskeletal dysfunction associated FHC mutations. Structural phenotypes specific to MLC2ventr/slow mutations in the heart will be correlated with the respective phenotypes in the slow-twitch skeletal muscles using small angle X-ray diffraction patterns. Histopathology and electron microscopy (EM) will complement the effect of mutations on structural reorganization of the sarcomere in the heart and soleus muscle. Measurements of contractile force, force-pCa relationship and the myosin cross-bridge kinetics in skinned papillary/soleus muscle fibers from all proposed Tg mouse models of FHC will complete the phenotypic characterization of MLC2ventr/slow-specific cardioskeletal myopathy. Importantly, we will also study the IVS6-1 mutation associated with premature infantile cardiac death.
AIM 2 : Determine FHC induced cardiac phenotypes in vivo and explore novel rescue mechanisms in transgenic mice expressing constitutively phosphorylated P-MLC2. We hypothesize that by altering the Ca2+-dependent regulation of muscle contraction, D166V and R58Q mutations increase the propensity of affected patients toward malignant disease phenotypes. We also hypothesize that the underlying mechanisms relate to the steric inhibition of myosin light chain kinase dependent phosphorylation of MLC2. These hypotheses will be addressed using our recently developed double mutant Tg-S15D-D166V rescue mice, designed to mitigate the effects of the malignant D166V mutation with a constitutively phosphorylated Ser-15 (S15D).

Public Health Relevance

Infantile fiber-type I hypotrophy with simultaneously occurring severe onset of cardiomyopathy was reported in multiple Dutch and Italian families and genetically linked to the MYL2 gene encoding for the human myosin regulatory light chain MLC2ventr/slow concurrently expressed in cardiac ventricles and in slow-twitch skeletal muscles. To address the molecular origin of this novel MLC2ventr/slow-induced cardioskeletal myopathy, this grant application will extensively investigate the functional consequences of familial hypertrophic cardiomyopathy (FHC)-linked mutations in MLC2 using cardiac papillary muscle fibers and slow-twitch skeletal soleus muscles from our developed transgenic (Tg) mice expressing MLC2 wild-type (Tg-WT) and FHC-linked mutants. Importantly, will also characterize the splice site IVS6-1 mutation in MYL2 shown to cause severe myopathy in humans and premature death of IVS6-1-homozygous patients. Together, this proposal will provide insight into molecular bases of FHC and a dual cardioskeletal myopathy caused by mutated MLC2ventr/slow.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL123255-01A1
Application #
8981118
Study Section
Special Emphasis Panel (ZRG1-MOSS-U (82))
Program Officer
Schramm, Charlene A
Project Start
2015-07-06
Project End
2019-04-30
Budget Start
2015-07-06
Budget End
2016-04-30
Support Year
1
Fiscal Year
2015
Total Cost
$542,614
Indirect Cost
$173,817
Name
University of Miami School of Medicine
Department
Pharmacology
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
Yadav, Sunil; Kazmierczak, Katarzyna; Liang, Jingsheng et al. (2018) Phosphomimetic-mediated in vitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain. FEBS J :
Yuan, Chen-Ching; Kazmierczak, Katarzyna; Liang, Jingsheng et al. (2018) Sarcomeric perturbations of myosin motors lead to dilated cardiomyopathy in genetically modified MYL2 mice. Proc Natl Acad Sci U S A 115:E2338-E2347
Wang, Li; Kazmierczak, Katarzyna; Yuan, Chen-Ching et al. (2017) Cardiac contractility, motor function, and cross-bridge kinetics in N47K-RLC mutant mice. FEBS J 284:1897-1913
Yadav, Sunil; Szczesna-Cordary, Danuta (2017) Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy. Biophys Rev 9:57-64
Yuan, Chen-Ching; Kazmierczak, Katarzyna; Liang, Jingsheng et al. (2017) Hypercontractile mutant of ventricular myosin essential light chain leads to disruption of sarcomeric structure and function and results in restrictive cardiomyopathy in mice. Cardiovasc Res 113:1124-1136
Zhou, Zhiqun; Huang, Wenrui; Liang, Jingsheng et al. (2016) Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants. Front Physiol 7:240
Wang, Yihua; Ajtai, Katalin; Kazmierczak, Katarzyna et al. (2016) N-Terminus of Cardiac Myosin Essential Light Chain Modulates Myosin Step-Size. Biochemistry 55:186-98
Szczesna-Cordary, Danuta; de Tombe, Pieter P (2016) Myosin light chain phosphorylation, novel targets to repair a broken heart? Cardiovasc Res 111:5-7
Huang, Wenrui; Kazmierczak, Katarzyna; Zhou, Zhiqun et al. (2016) Gene expression patterns in transgenic mouse models of hypertrophic cardiomyopathy caused by mutations in myosin regulatory light chain. Arch Biochem Biophys 601:121-32
Yuan, Chen-Ching; Muthu, Priya; Kazmierczak, Katarzyna et al. (2015) Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice. Proc Natl Acad Sci U S A 112:E4138-46

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