J.-P. Jin

Correcting diastolic dysfunction by Ca2+ desensitizing troponin in a transgenic mouse model of restrictive cardiomyopathy.

Several cardiac troponin I (cTnI) mutations are associated with restrictive cardiomyopathy (RCM) in humans. We have created transgenic mice (cTnI(193His) mice) that express the corresponding human RCM R192H mutation. Phenotype of this RCM animal model includes restrictive ventricles, biatrial enlargement and sudden cardiac death, which are similar to those observed in RCM patients carrying the same cTnI mutation. In the present study, we modified the overall cTnI in cardiac muscle by crossing cTnI(193His) mice with transgenic mice expressing an N-terminal truncated cTnI (cTnI-ND) that enhances relaxation. Protein analyses determined that wild type cTnI was replaced by cTnI-ND in the heart of double transgenic mice (Double TG), which express only cTnI-ND and cTnI R193H in cardiac myocytes. The presence of cTnI-ND effectively rescued the lethal phenotype of RCM mice by reducing the mortality rate. Cardiac function was significantly improved in Double TG mice when measured by echocardiography. The hypersensitivity to Ca(2+) and the prolonged relaxation of RCM cTnI(193His) cardiac myocytes were completely reversed by the presence of cTnI-ND in RCM hearts. The results demonstrate that myofibril hypersensitivity to Ca(2+) is a key mechanism that causes impaired relaxation in RCM cTnI mutant hearts and Ca(2+) desensitization by cTnI-ND can correct diastolic dysfunction and rescue the RCM phenotypes, suggesting that Ca(2+) desensitization in myofibrils is a therapeutic option for treatment of diastolic dysfunction without interventions directed at the systemic beta-adrenergic-PKA pathways.

Dose-dependent diastolic dysfunction and early death in a mouse model with cardiac troponin mutations

Our aim was to explore the dose-dependent diastolic dysfunction and the mechanisms of heart failure and early death in transgenic (TG) mice modeling human restrictive cardiomyopathy (RCM). The first RCM mouse model (cTnI(193His) mice) carrying cardiac troponin I (cTnI) R193H mutation (mouse cTnI R193H equals to human cTnI R192H) was generated several years ago in our laboratory. The RCM mice manifested a phenotype similar to that observed in RCM patients carrying the same cTnI mutation, i.e. enlarged atria and restricted ventricles. However, the causes of heart failure and early death observed in RCM mice remain unclear. In this study, we have produced RCM TG mice (cTnI(193His)-L, cTnI(193His)-M and cTnI(193His)-H) that express various levels of mutant cTnI in the heart. Histological examination and echocardiography were performed on these mice to monitor the time course of the disease development and heart failure. Our data demonstrate that cTnI mutation-caused diastolic dysfunction is dose-dependent. The key mechanism is myofibril hypersensitivity to Ca(2+) resulting in an impaired relaxation in the mutant cardiac myocytes. Prolonged relaxation time and delay of Ca(2+) decay observed in the mutant cardiac myocytes are correlated with the level of the mutant protein in the heart. Markedly enlarged atria due to the elevated end-diastolic pressure and myocardial ischemia are observed in the heart of the transgenic mice. In the mice with the highest level of the mutant protein, restricted ventricles and systolic dysfunction occur followed immediately by heart failure and early death. Diastolic dysfunction caused by R193H troponin I mutation is specific, showing a dose-dependent pattern. These mouse models are useful tools for the study of diastolic dysfunction. Impaired diastole can cause myocardial ischemia and fibrosis formation, resulting in the development of systolic dysfunction and heart failure with early death in the RCM mice with a high level of the mutant protein in the heart.

Up-regulation of alpha-smooth muscle actin in cardiomyocytes from non-hypertrophic and non-failing transgenic mouse hearts expressing N-terminal truncated cardiac troponin I ☆

We previously reported that a restrictive N‐terminal truncation of cardiac troponin I (cTnI‐ND) is up‐regulated in the heart in adaptation to hemodynamic stresses. Over‐expression of cTnI‐ND in the hearts of transgenic mice revealed functional benefits such as increased relaxation and myocardial compliance. In the present study, we investigated the subsequent effect on myocardial remodeling. The alpha‐smooth muscle actin (α‐SMA) isoform is normally expressed in differentiating cardiomyocytes and is a marker for myocardial hypertrophy in adult hearts. Our results show that in cTnI‐ND transgenic mice of between 2 and 3 months of age (young adults), a significant level of α‐SMA is expressed in the heart as compared with wild‐type animals. Although blood vessel density was increased in the cTnI‐ND heart, the mass of smooth muscle tissue did not correlate with the increased level of α‐SMA. Instead, immunocytochemical staining and Western blotting of protein extracts from isolated cardiomyocytes identified cardiomyocytes as the source of increased α‐SMA in cTnI‐ND hearts. We further found that while a portion of the up‐regulated α‐SMA protein was incorporated into the sarcomeric thin filaments, the majority of SMA protein was found outside of myofibrils. This distribution pattern suggests dual functions for the up‐regulated α‐SMA as both a contractile component to affect contractility and as possible effector of early remodeling in non‐hypertrophic, non‐failing cTnI‐ND hearts.

In Vivo Analysis of Troponin C Knock-In (A8V) Mice: Evidence that TNNC1 Is a Hypertrophic Cardiomyopathy Susceptibility Gene

Background— Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results— The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure–volume studies. Three-month-old KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V+/− mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography–mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+ sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+>KI-TnC-A8V+/−>wild-type, whereas KI-TnC-A8V+/+ relaxed more slowly on flash photolysis of diazo-2. Conclusions— The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.Background—Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results—The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure–volume studies. Three-month-old KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V+/− mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography–mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+ sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+>KI-TnC-A8V+/−>wild-type, whereas KI-TnC-A8V+/+ relaxed more slowly on flash photolysis of diazo-2. Conclusions—The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.

Restrictive cardiomyopathy mutations demonstrate functions of the C-terminal end-segment of troponin I.

The C-terminal end-segment of Troponin I (TnI) corresponding to the last 27-33 amino acids is the most conserved structure of TnI and interacts with tropomyosin in a Ca(2+)-regulated manner, suggesting a role in muscle relaxation. Mutations in the C-terminal end-segment of cardiac TnI cause restrictive cardiomyopathy. Here we demonstrate that mouse cardiac TnI containing R193H or R205H mutation have significantly conformational changes in the region interfacing with troponin T (TnT) and increased binding affinity for TnT. These restrictive cardiomyopathy mutations also exhibit increased binding affinity for troponin C at pCa 4. The effects of R193H mutation were more profound than that of R205H. Tertiary troponin complex was reconstituted using the TnI mutants and a mini TnT lacking tropomyosin-binding sites to examine the interaction between the C-terminal end-segment of TnI and tropomyosin. The results showed that, R193H, but not R205H, caused a moderate but statistically significant increase in the binding affinity for tropomyosin at pCa 9. Similar trend was observed at pCa 5.5 but not pCa 4. These results provide novel evidence for the function of the C-terminal end-segment of TnI, where mutations with conformational effects alter TnIs interaction with other troponin subunits and tropomyosin to cause diastolic dysfunction.

The heart-specific NH2-terminal extension regulates the molecular conformation and function of cardiac troponin I

In addition to the core structure conserved in all troponin I isoforms, cardiac troponin I (cTnI) has an ∼30 amino acids NH(2)-terminal extension. This peptide segment is a heart-specific regulatory structure containing two Ser residues that are substrates of PKA. Under β-adrenergic regulation, phosphorylation of cTnI in the NH(2)-terminal extension increases the rate of myocardial relaxation. The NH(2)-terminal extension of cTnI is also removable by restrictive proteolysis to produce functional adaptation to hemodynamic stresses. The molecular mechanism for the NH(2)-terminal modifications to regulate the function of cTnI is not fully understood. In the present study, we tested a hypothesis that the NH(2)-terminal extension functions by modulating the conformation of other regions of cTnI. Monoclonal antibody epitope analysis and protein binding experiments demonstrated that deletion of the NH(2)-terminal segment altered epitopic conformation in the middle, but not COOH-terminal, region of cTnI. PKA phosphorylation produced similar effects. This targeted long-range conformational modulation corresponded to changes in the binding affinities of cTnI for troponin T and for troponin C in a Ca(2+)-dependent manner. The data suggest that the NH(2)-terminal extension of cTnI regulates cardiac muscle function through modulating molecular conformation and function of the core structure of cTnI.

Localization and function of Xinα in mouse skeletal muscle

The Xin repeat-containing proteins were originally found in the intercalated discs of cardiac muscle with implicated roles in cardiac development and function. A pair of paralogous genes, Xinα (Xirp1) and Xinβ (Xirp2), is present in mammals. Ablation of the mouse Xinα (mXinα) did not affect heart development but caused late-onset adulthood cardiac hypertrophy and cardiomyopathy with conductive defects. Both mXinα and mXinβ are also found in the myotendinous junction (MTJ) of skeletal muscle. Here we investigated the structural and functional significance of mXinα in skeletal muscle. In addition to MTJ and the contact sites between muscle and perimysium, mXinα but not mXinβ was found in the blood vessel walls, whereas both proteins were absent in neuromuscular junctions and nerve fascicles. Coimmunoprecipitation suggested association of mXinα with talin, vinculin, and filamin, but not β-catenin, in adult skeletal muscle, consistent with our previous report of colocalization of mXinα with vinculin. Loss of mXinα in mXinα-null mice had subtle effects on the MTJ structure and the levels of several MTJ components. Diaphragm muscle of mXinα-null mice showed hypertrophy. Compared with wild-type controls, mouse extensor digitorum longus (EDL) muscle lacking mXinα exhibited no overt change in contractile and relaxation velocities or maximum force development but better tolerance to fatigue. Loaded fatigue contractions generated stretch injury in wild-type EDL muscle as indicated by a fragmentation of troponin T. This effect was blunted in mXinα-null EDL muscle. The results suggest that mXinα play a role in MTJ conductance of contractile and stretching forces.

Improved Fatigue Resistance in Gsα-Deficient and Aging Mouse Skeletal Muscles Due to Adaptive Increases in Slow Fibers

Genetically modified mice with deficiency of the G protein α-subunit (G(s)α) in skeletal muscle showed metabolic abnormality with reduced glucose tolerance, low muscle mass, and low contractile force, along with a fast-to-slow-fiber-type switch (Chen M, Feng HZ, Gupta D, Kelleher J, Dickerson KE, Wang J, Hunt D, Jou W, Gavrilova O, Jin JP, Weinstein LS. Am J Physiol Cell Physiol 296: C930-C940, 2009). Here we investigated a hypothesis that the switching to more slow fibers is an adaptive response with specific benefit. The results showed that, corresponding to the switch of myosin isoforms, the thin-filament regulatory proteins troponin T and troponin I both switched to their slow isoforms in the atrophic soleus muscle of 3-mo-old G(s)α-deficient mice. This fiber-type switch involving coordinated changes of both thick- and thin-myofilament proteins progressed in the G(s)α-deficient soleus muscles of 18- to 24-mo-old mice, as reflected by the expression of solely slow isoforms of myosin and troponin. Compared with age-matched controls, G(s)α-deficient soleus muscles with higher proportion of slow fibers exhibited slower contractile and relaxation kinetics and lower developed force, but significantly increased resistance to fatigue, followed by a better recovery. G(s)α-deficient soleus muscles of neonatal and 3-wk-old mice did not show the increase in slow fibers. Therefore, the fast-to-slow-fiber-type switch in G(s)α deficiency at older ages was likely an adaptive response. The benefit of higher fatigue resistance in adaption to metabolic deficiency and aging provides a mechanism to sustain skeletal muscle function in diabetic patients and elderly individuals.

Slow recovery of the impaired fatigue resistance in postunloading mouse soleus muscle corresponding to decreased mitochondrial function and a compensatory increase in type I slow fibers

Unloading or disuse rapidly results in skeletal muscle atrophy, switching to fast-type fibers, and decreased resistance to fatigue. The recovery process is of major importance in rehabilitation for various clinical conditions. Here we studied mouse soleus muscle during 60 days of reloading after 4 wk of hindlimb suspension. Unloading produced significant atrophy of soleus muscle with decreased contractile force and fatigue resistance, accompanied by switches of myosin isoforms from IIa to IIx and IIb and fast troponin T to more low-molecular-weight splice forms. The total mass, fiber size, and contractile force of soleus muscle recovered to control levels after 15 days of reloading. However, the fatigue resistance showed a trend of worsening during this period with significant infiltration of inflammatory cells at days 3 and 7, indicating reloading injuries that were accompanied by active regeneration with upregulations of filamin-C, αB-crystallin, and desmin. The fatigue resistance partially recovered after 30-60 days of reloading. The expression of peroxisome proliferator-activated receptor γ coactivator 1α and mitofusin-2 showed changes parallel to that of fatigue resistance after unloading and during reloading, suggesting a causal role of decreased mitochondrial function. Slow fiber contents in the soleus muscle were increased after 30-60 days of reloading to become significantly higher than the normal level, indicating a secondary adaption to compensate for the slow recovery of fatigue resistance.

Deletion of calponin 2 attenuates the development of calcific aortic valve disease in ApoE−/− mice

Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular mortality and lacks non-surgical treatment. The pathogenesis of CAVD involves perturbation of valvular cells by mechanical stimuli, including shear stress, pressure load and leaflet stretch, of which the molecular mechanism requires further elucidation. We recently demonstrated that knockout (KO) of Cnn2 gene that encodes calponin isoform 2, a mechanoregulated cytoskeleton protein, attenuates atherosclerosis in ApoE KO mice. Here we report that Cnn2 KO also decreased calcification of the aortic valve in ApoE KO mice, an established model of CAVD. Although myeloid cell-specific Cnn2 KO highly effectively attenuated vascular atherosclerosis that shares many pathogenic processes with CAVD, it did not reduce aortic valve calcification in ApoE KO mice. Indicating a function in the pathogenesis of CAVD, calponin 2 participates in myofibroblast differentiation that is a leading step in the development of CAVD. The aortic valves of ApoE KO mice exhibited increased expression of calponin 2 and smooth muscle actin (SMA), a hallmark of myofibroblasts. The expression of calponin 2 increased during myofibroblast-like differentiation of primary sheep aortic valve interstitial cells and during the osteogenic differentiation of mouse myofibroblasts. Cnn2 KO attenuated TGFβ1-induced differentiation of myofibroblasts in culture as shown by the lower expression of SMA and less calcification than that of wild type (WT) cells. These findings present calponin 2 as a novel molecular target for the treatment and prevention of CAVD.