The Effects Of SM22α Knockout On Cardiac Function And Its Underlying Mechanisms In Mice

OBJECTIVES:SM22α is an actin binding protein that promotes gelation of actin filaments.Recently,multiple lines of evidence suggest that SM22α is involved in regulation of tension sensing in smooth muscle cells(SMCs).Increasing evidence suggests that specialized membrane structure,caveolae and T-Tubules,are emerging as essential components in mechanotransduction in cardiomyocytes.Here we investigated the effects of SM22α knockout on cardiac function and its underlying mechanisms in mice.METHODS:SM22α knockout(SM22α KO)mice were utilized in this study to assess the role of SM22α in cardiomyocytes.Transverse aortic constriction(TAC)was conducted to induce heart failure,and echocardiography was used to assess cardiac function.Cell shortening properties were measured by IonOptix devices in intact cardiomyocytes,calcium sensitivity of myofilament was measured in intact and saponin permeabilized cardiomyocytes.Confocal microscopy,transmission electron microscopy(TEM),western blotting,immunoprecipitation,RT-PCR techniques were used to perform functional and structural analysis of SM22αin cardiomyocytes.RESULTS:1.Disruption of SM22a results in decreased heart functions in mice1.1 SM22α exists in cardiomyocytes of adult miceIt is still under debate whether SM22α exists in the heart growing into adulthood.Here,we showed the expression of SM22α protein and mRNA in both cardiac tissues and enzymatically isolated cardiomyocytes of mice.1.2 SM22α deletion impairs cardiac function in miceAlthough SM22α is not required for the development and basal homeostatic function of VSMCs in the developing mouse,lack of SM22αresulted in a subtle but significant decrease in ejection fraction in mice under basal conditions(52.805 ± 0.658%,n=162)compared with wild-type(WT)controls(55.469 ± 0.611%,n=145,P<0.01).The mRNA levels of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and β-myosin heavy chain(β-MHC)were significantly increased in SM22α KO hearts over controls,indicating the SM22α KO mice showed a tendency toward decreased cardiac function even under basal conditions.1.3 Upon pressure overloading induced by transverse aortic constriction(TAC),the SM22α KO mice developed into heart failure quicker than WT miceTo assess the role of SM22α in the heart,SM22α KO and WT mice were challenged with pressure overload induced by TAC.SM22α KO mice subjected to TAC for 2 weeks developed profound cardiac dysfunction which was characterized as reduction in ejection fraction 48.42 ± 1.44%(n=35,P<0.01 vs.SM22α KO control;P<0.001 vs.WT),whereas the WT animals were able to sustain a compensated state.By 4 weeks,mice were killed,and hearts were excised.Hearts from SM22α KO mice subjected to TAC were enlarged compared with WT controls,and their lung weight/body weight ratio and heart weight/body weight ratio were significantly increased compared with WT controls.1.4 Increased SM22α in heart post 4 weeks of WT heartNext we detected the expression level of SM22α in WT cardiomyocytes 4 weeks upon TAC.The protein level of SM22α was increased in WT cardiomyocytes post TAC(Sham:0.36 ± 0.05,n=5;TAC:0.63 ± 0.09,n=5;P<0.05).The mRNA level of SM22α was 60%increased(Sham:1.00 ± 0.10,n=21;TAC:1.63 ± 0.14,n=12,P<0.001).2.Deletion of SM22α impairs the structure and function of caveolae in cardiomyocytes2.1 Caveolae are disassembled in ventricular myocytes of SM22α KO miceTo elucidate the possible connection between SM22α and caveolae structure,we examined caveolar abundance in WT and SM22α KO hearts by transmission electron microscopy.The density of caveolae on the plasma membrane of ventricular myocytes was significantly reduced in SM22α KO mice relative to WT hearts under basal condition.Moreover,the morphology of individual caveolae was strikingly altered under basal condition already.The classic flask-shape or omega-shape invagination disappeared,and the opening width of caveolae was more than doubled with the maximum width and the depth of caveolae being comparable in KO and control animals.This marked change may have a potentially significant impact on the ability of the sarcolemmal membrane of cardiomyocytes to buffer the increased stress following TAC.2.2 SM22α ablation disrupted the interaction between actin and caveolin-3 in cardiomyocyteCaveolin-3(Cav3)is a major constituent of cardiac caveolae.We showed that the expression of Cav3 in SM22α KO heart is comparable to that in WT control,suggesting the above mentioned morphological alterations in caveolae is independent to CAV3 expression level.Caveolae and caveolin-associated signaling proteins and receptors are linked via structural proteins to a dynamic filamentous actin network.Next we detected the interaction among Cav3,α-actin and SM22α in the myocytes using co-immunoprecipitation.CAV3 can be co-immunoprecipitated with anti-SM22α antibody and vice versa,suggesting binding between SM22α and CAV3.Additionally,we found the amount of actin associated with CAV3 is reduced in SM22α KO mice.2.3 Altered distribution of Tcap in SM22α KO ventricular myocytesTelethonin(Tcap)is a 19 kDa Z-disk protein in cardiac muscle and skeletal muscle,which assembled in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing.Next we detected the expression and distribution of Tcap in cardiomyocytes of SM22α KO mice.Although we showed the there is no difference of the expression level between two genotypes,we found the nuclear accumulation of Tcap in SM22α KO cardiomyocytes.2.4 SM22α ablation alters kinetics of cell shorteningKinetics of cell shortening were measured by IonOptix devices.The amplitude of cell shortening was reduced by approximately 10%in SM22αKO mice(3.843 ± 0.363%)compared with controls(4.354 ± 0.201%),although the difference was statistically insignificant(P=0.059),most kinetic properties are significantly altered in knockout mice,such as time to peak(WT:0.080 ± 0.004;SM22α KO:0.059 ± 0.003,P<0.001)and max contraction velocity(WT:1.455 ± 0.123;SM22α KO:1.147 ± 0.077,P<0.05).These data suggested that the kinetics of sarcomere shortening was altered in knockout mice.2.5 Calcium sensitivity of myofilament is increased in SM22α KO miceTo assess whether the change in contraction kinetics is a direct effect or not,we permeabilized the plasma membrane of cardiomyocytes with saponin and perfused the cells with the same set of intracellular buffers,and with different free calcium concentrations to measure the calcium sensitivity of myofilament.We found the max response was reduced in the SM22α KO mice with increased calcium sensitivity.Our data suggest that the myocytes missing SM22α may be more sensitive to lower calcium concentration,and difficult to meet the need of the heart at increased calcium concentrations.3.SM22α ablation disrupts t-tubules regularity of ventricular myocytes by reduction of JPH2 expression3.1 Disrupted T-Tubules in ventricular myocytes of SM22α KO miceMembrane invaginations called t-tubules play an integral role in triggering cardiomyocyte con-traction,and their disruption are believed to be a key cause of heart failure.Next we stained plasma membrane with Di-8-ANEPPS and perform confocal microscopy to evaluate the integrity of T-Tubules in SM22α KO mice.We showed that the well-organized t-tubule network observed in myocytes from WT hearts is lost and disorganized in the cells of SM22α KO mice.Quantification of t-tubule organization using TT power and TT score confirmed that t-tubule regularity was significantly reduced in SM22α KO mice.Junctophilin-2(JPH2)is critical in anchoring the invaginating sarcolemma to the sarcoplasmic reticulum,thereby enabling the maturation of the t-tubule network.Transcriptomic analysis of arteries of SM22α KO mice revealed a significant reduction in JPH2 mRNA level in VSMCs.Similarly,the expression of JPH2 in cardiac tissue reduced by 30%～60%at mRNA and protein levels,respectively.Furthermore,JPH2 interacted with SM22α that was immunoprecipitated with JPH2 antibody.These findings suggest that SM22α may be involved in the maturation of t-tubules via recruitment of JPH23.2 Spatial synchronization of calcium transient is reduced by SM22α KOUsually disrupted T-Tubules will lead to asynchronized SR calcium release following depolarization.Next we examined calcium transients in both WT and SM22α KO cardiomyocytes with con-focal microscopy in line-scan mode.The amplitude of calcium transients amplitude and calcium decay constant(τ)were comparable between WT and SM22α KO myocytes,whereas the time to peak(TtP)was significantly prolonged in SM22α KO mice,suggesting asynchronized SR calcium release throughout the cell due to disrupted T-Tubules.3.3 Intracellular calcium homeostasis is disrupted in SM22α KO miceThe synchronization of SR Ca2+release depends on the mature t-tubule network.Next we test whether T-tubule disruption correlated with defective calcium release.We used confocal line scan imaging to visualize Ca2+sparks in isolated cardiomyocytes.Knockout of SM22a led to a significantly increased Ca2+spark frequency,suggesting hyperactivity of RyR2 and enhanced RyR2-mediated SR calcium leak.Therefore next we assessed SR Ca2+content with a caffeine dump protocol.We found the SR calcium content was significantly decreased in SM22α KO mice.CONCLUSION:Adult SM22α KO mice exhibit less and deformed caveolae and T-Tubules in cardiomyocytes.Loss of SM22α leads to impaired contractile properties and altered membrane structure of cardiac myocytes,and thereby causing the heart more vulnerable to animal models of cardiac stress.Our findings,identifying a novel role for SM22α,provide insight into the mechanisms underlying caveolae-and T-Tubules-mediated adaptation to cardiac stress.