Mechano-growth factor E-domain modulates cardiac contractile function through 14-3-3 protein interactomes.

In the heart, alternative splicing of the igf-I gene produces two isoforms: IGF-IEa and IGF-IEc, (Mechano-growth factor, MGF). The sequence divergence between their E-domain regions suggests differential isoform function. To define the biological actions of MGF's E-domain, we performed in silico analysis of the unique C-terminal sequence and identified a phosphorylation consensus site residing within a putative 14-3-3 binding motif. To test the functional significance of Ser 18 phosphorylation, phospho-mimetic (S/E18) and phospho-null (S/A18) peptides were delivered to mice at different doses for 2 weeks. Cardiovascular function was measured using echocardiography and a pressure-volume catheter. At the lowest (2.25 mg/kg/day) and highest (9 mg/kg/day) doses, the peptides produced a depression in systolic and diastolic parameters. However, at 4.5 mg/kg/day the peptides produced opposing effects on cardiac function. Fractional shortening analysis also showed a similar trend, but with no significant change in cardiac geometry. Microarray analysis discovered 21 genes (FDR p < 0.01), that were expressed accordant with the opposing effects on contractile function at 4.5 mg/kg/day, with the nuclear receptor subfamily 4 group A member 2 (Nr4a2) identified as a potential target of peptide regulation. Testing the regulation of the Nr4a family, showed the E-domain peptides modulate Nr4a gene expression following membrane depolarization with KCl in vitro. To determine the potential role of 14-3-3 proteins, we examined 14-3-3 isoform expression and distribution. 14-3-3γ localized to the myofilaments in neonatal cardiac myocytes, the cardiac myocytes and myofilament extracts from the adult heart. Thermal shift analysis of recombinant 14-3-3γ protein showed the S/A18 peptide destabilized 14-3-3γ folding. Also, the S/A18 peptide significantly inhibited 14-3-3γ's ability to interact with myosin binding protein C (MYPC3) and phospholamban (PLN) in heart lysates from dobutamine injected mice. Conversely, the S/E18 peptide showed no effect on 14-3-3γ stability, did not inhibit 14-3-3γ's interaction with PLN but did inhibit the interaction with MYPC3. Replacing the glutamic acid with a phosphate group on Ser 18 (pSer18), significantly increased 14-3-3γ protein stability. We conclude that the state of Ser 18 phosphorylation within the 14-3-3 binding motif of MGF's E-domain, modulates protein-protein interactions within the 14-3-3γ interactome, which includes proteins involved in the regulation of contractile function.

Localized delivery of mechano-growth factor E-domain peptide via polymeric microstructures improves cardiac function following myocardial infarction.

The Insulin like growth factor-I isoform mechano-growth factor (MGF), is expressed in the heart following myocardial infarction and encodes a unique E-domain region. To examine E-domain function, we delivered a synthetic peptide corresponding to the unique E-domain region of the human MGF (IGF-1Ec) via peptide eluting polymeric microstructures to the heart. The microstructures were made of poly (ethylene glycol) dimethacrylate hydrogel and bioengineered to be the same size as an adult cardiac myocyte (100 × 15 × 15 µm) and with a stiffness of 20 kPa. Peptide eluting microrods and empty microrods were delivered via intramuscular injection following coronary artery ligation in mice. To examine the physiologic consequences, we assessed the impact of peptide delivery on cardiac function and cardiovascular hemodynamics using pressure-volume loops and gene expression by quantitative RT-PCR. A significant decline in both systolic and diastolic function accompanied by pathologic hypertrophy occurred by 2 weeks which decompensated further by 10 weeks post-infarct in the untreated groups. Delivery of the E-domain peptide eluting microrods decreased mortality, ameliorated the decline in hemodynamics, and delayed decompensation. This was associated with the inhibition of pathologic hypertrophy despite increasing vascular impedance. Delivery of the empty microrods had limited effects on hemodynamics and while pathologic hypertrophy persisted there was a decrease in ventricular stiffness. Our data show that cardiac restricted administration of the MGF E-domain peptide using polymeric microstructures may be used to prevent adverse remodeling of the heart and improve function following myocardial infarction.

Microarray analysis of active cardiac remodeling genes in a familial hypertrophic cardiomyopathy mouse model rescued by a phospholamban knockout.

Familial hypertrophic cardiomyopathy (FHC) is a disease characterized by ventricular hypertrophy, fibrosis, and aberrant systolic and/or diastolic function. Our laboratories have previously developed two mouse models that affect cardiac performance. One mouse model encodes an FHC-associated mutation in α-tropomyosin: Glu → Gly at amino acid 180, designated as Tm180. These mice display a phenotype that is characteristic of FHC, including severe cardiac hypertrophy with fibrosis and impaired physiological performance. The other model was a gene knockout of phospholamban (PLN KO), a regulator of calcium uptake in the sarcoplasmic reticulum of cardiomyocytes; these hearts exhibit hypercontractility with no pathological abnormalities. Previous work in our laboratories shows that when mice were genetically crossed between the PLN KO and Tm180, the progeny (PLN KO/Tm180) display a rescued hypertrophic phenotype with improved morphology and cardiac function. To understand the changes in gene expression that occur in these models undergoing cardiac remodeling (Tm180, PLN KO, PLN KO/Tm180, and nontransgenic control mice), we conducted microarray analyses of left ventricular tissue at 4 and 12 mo of age. Expression profiling reveals that 1,187 genes changed expression in direct response to the three genetic models. With these 1,187 genes, 11 clusters emerged showing normalization of transcript expression in the PLN KO/Tm180 hearts. In addition, 62 transcripts are highly involved in suppression of the hypertrophic phenotype. Confirmation of the microarray analysis was conducted by quantitative RT-PCR. These results provide insight into genes that alter expression during cardiac remodeling and are active during modulation of the cardiomyopathic phenotype.

Long-term rescue of a familial hypertrophic cardiomyopathy caused by a mutation in the thin filament protein, tropomyosin, via modulation of a calcium cycling protein.

We have recently shown that a temporary increase in sarcoplasmic reticulum (SR) cycling via adenovirus-mediated overexpression of sarcoplasmic reticulum ATPase (SERCA2) transiently improves relaxation and delays hypertrophic remodeling in a familial hypertrophic cardiomyopathy (FHC) caused by a mutation in the thin filament protein, tropomyosin (i.e., α-TmE180G or Tm180). In this study, we sought to permanently alter calcium fluxes via phospholamban (PLN) gene deletion in Tm180 mice in order to sustain long-term improvements in cardiac function and adverse cardiac remodeling/hypertrophy. While similar work has been done in FHCs resulting from mutations in thick myofilament proteins, no one has studied these effects in an FHC resulting from a thin filament protein mutation. Tm180 transgenic (TG) mice were crossbred with PLN knockout (KO) mice and four groups were studied in parallel: 1) non-TG (NTG), 2) Tm180, 3) PLNKO/NTG and 4) PLNKO/Tm180. Tm180 mice exhibit increased heart weight/body weight and hypertrophic gene markers compared to NTG mice, but levels in PLNKO/Tm180 mice were similar to NTG. Tm180 mice also displayed altered function as assessed via in situ pressure-volume analysis and echocardiography at 3-6 months and one year; however, altered function in Tm180 mice was rescued back to NTG levels in PLNKO/Tm180 mice. Collagen deposition, as assessed by Picrosirius Red staining, was increased in Tm180 mice but was similar in NTG and in PLNKO/Tm180 mice. Extracellular signal-regulated kinase (ERK1/2) phosphorylation increased in Tm180 mice while levels in PLNKO/Tm180 mice were similar to NTGs. The present study shows that by modulating SR calcium cycling, we were able to rescue many of the deleterious aspects of FHC caused by a mutation in the thin filament protein, Tm.

Neonatal gene transfer of Serca2a delays onset of hypertrophic remodeling and improves function in familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant genetic disorder linked to numerous mutations in the sarcomeric proteins. The clinical presentation of FHC is highly variable, but it is a major cause of sudden cardiac death in young adults with no specific treatments. We tested the hypothesis that early intervention in Ca(2+) regulation may prevent pathological hypertrophy and improve cardiac function in a FHC displaying increased myofilament sensitivity to Ca(2+) and diastolic dysfunction. A transgenic (TG) mouse model of FHC with a mutation in tropomyosin at position 180 was employed. Adenoviral-Serca2a (Ad.Ser) was injected into the left ventricle of 1-day-old non-transgenic (NTG) and TG mice. Ad.LacZ was injected as a control. Serca2a protein expression was significantly increased in NTG and TG hearts injected with Ad.Ser for up to 6 weeks. Compared to TG-Ad.LacZ hearts, the TG-Ad.Ser hearts showed improved whole heart morphology. Moreover, there was a significant decline in ANF and ß-MHC expression. Developed force in isolated papillary muscle from 2- to 3-week-old TG-Ad.Ser hearts was higher and the response to isoproterenol (ISO) improved compared to TG-Ad.LacZ muscles. In situ hemodynamic measurements showed that by 3 months the TG-Ad.Ser hearts also had a significantly improved response to ISO compared to TG-Ad.LacZ hearts. The present study strongly suggests that Serca2a expression should be considered as a potential target for gene therapy in FHC. Moreover, our data imply that development of FHC can be successfully delayed if therapies are started shortly after birth.

Ablation of iNOS delays cardiac contractile dysfunction in chronic hypertension.

We investigated the role of inducible NOS (iNOS) on cardiac function during the development of left ventricular hypertrophy. Hypertrophy was induced by pressure-overload via short-term (2.5 months) or long-term (6.5 months) aortic banding (AoB) in wild-type (WT) and iNOS knock out (iNOSKO) mice. Cardiac function was then assessed via echocardiography, in situ hemodynamics and papillary muscle force measurements. Quantitative RT-PCR and Western blots were used to measure expression of hypertrophic gene markers and proteins respectively. Our data demonstrate that increased afterload via AoB leads to increased expression of iNOS that is associated with cardiac dysfunction. In pressure-overload induced hypertrophy, iNOSKO delays both the expression of hypertrophic markers and contractile dysfunction without causing significant changes in the level of hypertrophy. Moreover, after long-term AoB, iNOSKO animals exhibited increased basal cardiac function and an improved response to beta-adrenergic stimulation compared to long-term AoB WT animals. In conclusion, our data demonstrate that NO production via iNOS plays an important role in modulating cardiac function after moderate AoB that mimics long-term hypertension in humans.

Myofilament response to Ca2+ and Na+/H+ exchanger activity in sex hormone-related protection of cardiac myocytes from deactivation in hypercapnic acidosis.

Compared to sham-operated controls, myofilaments from hearts of ovariectomized (OVX) rats demonstrate an increase in Ca2+ sensitivity with no change in maximum tension (Wattanapermpool J and Reiser PJ. Am J Physiol 277: H467-H473, 1999). To test the significance of this modification in intact cells, we compared intracellular Ca2+ transients and shortening of ventricular myocytes isolated from sham and 10-wk OVX rats. There was a decrease in the peak Ca2+ transient with prolonged 50% decay time in OVX cardiac myocytes without changes in the resting intracellular Ca2+ concentration. Percent cell shortening was also depressed, and relaxation was prolonged in cardiac myocytes from OVX rats compared with shams. Ovariectomy induced a sensitization of the myofilaments to Ca2+. Hypercapnic acidosis suppressed the shortening of OVX myocytes to a lesser extent than that detected in shams. Moreover, a larger compensatory increase in %cell shortening was obtained in OVX myocytes during prolonged acidosis. The elevated compensation in cell shortening was related to a higher amount of increase in the amplitude of the Ca2+ transient in OVX myocytes. However, these differences in Ca2+ transients and %cell shortening were no longer evident in the presence of 1 microM cariporide, a specific inhibitor of Na+/H+ exchanger type 1 (NHE1). Our results indicate that deprivation of female sex hormones modulates the intracellular Ca2+ concentration in cardiac myocytes, possibly via an increased NHE1 activity, which may act in concert with Ca2+ hypersensitivity of myofilament activation as a determinant of sex differences in cardiac function.

Correlations between alterations in length-dependent Ca2+ activation of cardiac myofilaments and the end-systolic pressure-volume relation.

We have tested the hypothesis that alterations in length dependent activation (LDA) of cardiac myofilaments represent an important regulatory mechanism affecting the Frank-Starling mechanism as determined by the slope (E(es)) of the relation between left ventricular (LV) volume and end-systolic pressure. We employed a transgenic (TG) mouse model in which the cardiac isoform of TnI (cTnI) has been completely replaced with slow skeletal TnI (ssTnI), the embryonic/neonatal isoform in the heart. Compared to non-transgenic (NTG) controls, myofilaments from TG-ssTnI hearts demonstrate an increase in Ca(2+) sensitivity and a substantially blunted LDA that is unaffected by PKA-dependent phosphorylation. We measured in situ LV pressure and volume relations during basal conditions and isoproterenol (ISO) stimulation. In the basal state in TG-ssTnI hearts there was significant increase in end-systolic pressure and slight decrease in heart rate. ISO stimulation resulted in a significant increase in heart rate, ejection fraction, maximum dP/dt, preload-recruitable stroke work, maximum dP/dt versus end diastolic volume and cardiac output in both groups. During basal conditions there was no difference in the E(es) relation between NTG and TG-ssTnI groups. However, during ISO stimulation the E(es) relation was significantly different between NTG and TG-ssTnI groups. Our study provides the first direct evidence that enhancement in differences in LDA between cardiac myofilaments from NTG and TG-ssTnI hearts induced by post-translational modifications of sarcomeric proteins are reflected in the in situ beating heart by a different change in E(es). Thus, changes in LDA should be considered in interpreting results from in situ experiments on inotropic effects associated with physiological and patho-physiological states of the heart.

The effect of myosin regulatory light chain phosphorylation on the frequency-dependent regulation of cardiac function.

Although it has been suggested that in cardiac muscle the phosphorylation level of myosin regulatory light chain (RLC) correlates with frequency of stimulation, its significance in the modulation of the force-frequency and pressure-frequency relationships remains unclear. We examined the role of RLC phosphorylation on the force-frequency relation (papillary muscles), the pressure-frequency relation (Langendorff perfused hearts) and shortening-frequency relation (isolated cardiac myocytes) in nontransgenic (NTG) and transgenic mouse hearts expressing a nonphosphorylatable RLC protein (RLC(P-)). At 22 degrees C, NTG and RLC(P-) muscles showed a negative force-frequency relation. At 32 degrees C, at frequencies above 1 Hz, both groups showed a flat force-frequency relation. There was a small increase in RLC phosphorylation in NTG muscles when the frequency of stimulation was increased from 0.2 Hz to 4.0 Hz. However, the level of RLC phosphorylation in these isolated muscles was significantly lower compared to samples taken from NTG intact hearts. In perfused hearts, there was no difference in the slope of pressure-frequency relationship between groups, but the RLC(P-) group consistently developed a reduced systolic pressure and demonstrated a decreased contractility. There was no difference in the level of RLC phosphorylation in hearts paced at 300 and 600 bpm. In RLC(P-) hearts, the level of TnI phosphorylation was reduced compared to NTG. There was no change in the expression of PLB between groups, but expression of SERCA2 was increased in hearts from RLC(P-) compared to NTG. In isolated cardiac myocytes, there was no change in shortening-frequency relationship between groups. Moreover, there was no change in Ca(2+) transient parameters in cells from NTG and RLC(P-) hearts. Our data demonstrate that in cardiac muscle RLC phosphorylation is not an essential determinant of force- and pressure-frequency relations but the absence of RLC phosphorylation decreases contractility in force/pressure developing preparations.

Expression of slow skeletal troponin I in adult mouse heart helps to maintain the left ventricular systolic function during respiratory hypercapnia.

Compared with the adult, neonatal heart muscle is less sensitive to deactivation by acidic pH. We hypothesized that expression of slow skeletal troponin I (ssTnI), the embryonic isoform, in adult heart would help maintain left ventricular (LV) systolic function during respiratory hypercapnia. We assessed LV function by transthoracic 2D-targeted M-mode and pulsed Doppler echocardiography in transgenic (TG) mice in which cardiac TnI was replaced with ssTnI and in nontransgenic (NTG) littermates. Anesthetized mice were ventilated with either 100% oxygen or 35% CO2 balanced with oxygen. Arterial blood pH with 35% CO2 decreased to the same levels in both groups of animals. In the absence of propranolol, the LV fractional shortening was higher in TG compared with NTG mice throughout most of the experimental protocol. LV diastolic function was impaired in TG compared with NTG mice both at 100% oxygen and 35% CO2 because E-to-A wave ratio of mitral flow was significantly lower, and E-wave deceleration time and LV isovolumic relaxation time were longer in TG compared with NTG mice. When compensatory mechanisms that occur through stimulation of beta-adrenergic receptors during hypercapnia were blocked by continuous perfusion with propranolol, we found that NTG mice died within 3 to 4 minutes after switching to 35% CO2, whereas TG mice survived. Our experiments demonstrate the first evidence that specific replacement of cardiac TnI with ssTnI has a protective effect on the LV systolic function during hypercapnic acidosis in situ.

Differential effects of isoflurane and ketamine/inactin anesthesia on cAMP and cardiac function in FVB/N mice during basal state and beta-adrenergic stimulation.

We evaluated the effect of the inhalant anesthetic isoflurane and the injectable combination of anesthetics ketamine/inactin on cardiac function by measuring left ventricular (LV) pressure in situ during control conditions and during beta-adrenergic stimulation with isoproterenol (ISO). The control heart rate (HR) and the maximal rate of contraction were significantly higher in the isoflurane group, but there was no difference in the rate of relaxation. During the ISO (0.32 ng g body wt(-1) min(-1)) stimulation the developed pressure (DP) increased 9.8 +/- 1.8% (n = 11) in the ketamine/inactin group and was unchanged in the isoflurane group. The HR increased 28.4 +/- 4.8% (n = 11) in the ketamine/inactin group and only 3.4 +/- 0.6% (n = 11) in the isoflurane group. The rate of contraction increased 103.2 +/- 9.3% (n = 11) and 13.6 +/- 4.6% (n = 11) in the ketamine/inactin and isoflurane groups, respectively. At this dose of ISO the rate of relaxation did not change significantly. In control conditions there was no difference in levels of cAMP between the groups (2.29 +/- 0.25 pmol/mg protein (n = 5) in the ketamine/inactin group and 2.79 +/- 0.35 pmol/mg protein (n = 6) in the isoflurane group). However, during the ISO stimulation the cAMP level increased only in the ketamine/ inactin group of animals (3.50 +/- 0.30 pmol/mg protein; n = 5). This level was significantly higher than the level in the isoflurane group stimulated with ISO (2.22 +/- 0.30 pmol/mg protein; n = 6). In summary, our results indicate that the anesthetics differ significantly in the extent of depression of the basal and beta-adrenergic stimulated state with the second messenger cAMP playing a prominent role.

Troponin I phosphorylation plays an important role in the relaxant effect of beta-adrenergic stimulation in mouse hearts.

OBJECTIVE: The present study was designed to address the question of the contribution of cardiac troponin I (cTnI) phosphorylation to the enhanced rate of relaxation during beta-adrenergic stimulation in hearts in situ. METHODS: In situ hemodynamic measurements were performed in mouse hearts that (1) express normal level of phospholamban (PLB) and either express cTnI (PLB/cTnI) or the slow skeletal isoform of TnI (PLB/ssTnI) that cannot be phosphorylated by protein kinase A (PKA) or (2) do not express PLB and either express cTnI (PLBKO/cTnI) or ssTnI (PLBKO/ssTnI). RESULTS: In the basal state, there was no difference in heart rate (HR), developed pressure (DP), left ventricular end-diastolic pressure (LVEDP) or rate of contraction (+dP/dt) between PLB/cTnI and PLB/ssTnI groups. However, hearts expressing ssTnI (PLB/ssTnI) showed a significantly decreased rate of relaxation (-dP/dt) when compared with hearts expressing cTnI (PLB/cTnI). In response to beta-adrenergic agonist, isoproterenol (ISO), HR increased similarly in both groups. At the two highest doses of ISO, the rate of relaxation (-dP/dt) was significantly smaller in PLB/ssTnI than in PLB/cTnI hearts. In the basal state, there was no difference in HR, DP, LVEDP,+dP/dt and -dP/dt between PLBKO/cTnI and PLBKO/ssTnI hearts. In response to ISO, HR increased similarly in both groups and was only slightly smaller in PLBKO/ssTnI group at the lowest dose of ISO. However, during ISO perfusion, when cTnI was phosphorylated, the rate of relaxation was significantly slower in PLBKO/ssTnI compared to PLBKO/cTnI hearts. CONCLUSION: Our data support the hypothesis that phosphorylation of cTnI significantly contributes to the enhanced rate of relaxation during beta-adrenergic stimulation.

Expression of slow skeletal troponin I in hearts of phospholamban knockout mice alters the relaxant effect of beta-adrenergic stimulation.

Beta-adrenergic stimulation of the heart results in an enhanced relaxation rate in association with phosphorylation of both cardiac troponin I (cTnI) and phospholamban (PLB). We studied new lines of mice generated by crossbreeding mice that express slow skeletal troponin I (ssTnI) with PLB knockout (PLBKO) mice. This crossbreeding resulted in the generation of PLB/cTnI, PLB/ssTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. Perfusion with isoproterenol (ISO) significantly increased the peak amplitude of fura-2 ratio in PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI groups of mice. However, in the presence of ISO, there were no differences in the peak amplitude of fura-2 ratio among cells isolated from hearts of PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. In cells from PLB/cTnI mice, the extent of shortening was increased and the time of relaxation was significantly decreased during beta-adrenergic stimulation. In PLBKO/cTnI cells, stimulation with ISO resulted in an increased extent of shortening and no change in time of relaxation. However, stimulation with ISO in cells isolated from PLBKO/ssTnI mice not only significantly increased the extent of cell shortening but also increased the time of relaxation. We also determined the kinetics of relaxation of papillary muscles isolated from all four groups of animals in the presence and absence of ISO. Perfusion with ISO increased the rate of relaxation only in PLB/cTnI, PLB/ssTnI, and PLBKO/cTnI muscles. During ISO stimulation, the time of relaxation was unchanged in PLBKO/ssTnI muscles. Our data directly demonstrate that phosphorylation of both PLB and cTnI contributes to increased rate of relaxation during beta-adrenergic stimulation.