Effects of nicotine administration in a mouse model of familial hypertrophic cardiomyopathy, α-tropomyosin D175N

The effects of nicotine (NIC) on normal hearts are fairly well established, yet its effects on hearts displaying familial hypertrophic cardiomyopathy have not been tested. We studied both the acute and chronic effects of NIC on a transgenic (TG) mouse model of FHC caused by a mutation in α-tropomyosin (Tm; i.e., α-Tm D175N TG, or Tm175). For acute effects, intravenously injected NIC increased heart rate, left ventricular (LV) pressure, and the maximal rate of LV pressure increase (+dP/dt) in non-TG (NTG) and Tm175 mice; however, Tm175 showed a significantly smaller increase in the maximal rate of LV pressure decrease (-dP/dt) compared with NTGs. Western blots revealed phosphorylation of phospholamban Ser16 and Thr17 residue increased in NTG mice following NIC injection but not in Tm175 mice. In contrast, phosphorylation of troponin I at serine residues 23 and 24 increased equally in both NTG and Tm175. Thus the attenuated increase in relaxation in Tm175 mice following acute NIC appears to result primarily from attenuated phospholamban phosphorylation. Chronic NIC administration (equivalent to smoking 2 packs of cigarettes/day for 4 mo) also increased +dP/dt in NTG and Tm175 mice compared with chronic saline. However, chronic NIC had little effect on heart rate, LV pressure, -dP/dt, LV wall and chamber dimensions, or collagen content for either group of mice.     

Targeted inhibition of sarcoplasmic reticulum CaMKII activity results in alterations of Ca2+ homeostasis and cardiac contractility

Transgenic (TG) mice expressing a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr17 and develop dilated myopathy when stressed by gestation and parturition (Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. J Biol Chem 278: 25063-25071, 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of sarcoplasmic reticulum (SR) CaMKII activity on the contractile characteristics and Ca2+ cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (+/-dP/dt) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild-type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40-47% decrease in the maximal rates of myocyte shortening and relengthening under both basal and Iso-stimulated conditions. Although twitch Ca2+ transient amplitudes were not significantly altered, the rate of twitch intracellular Ca2+ concentration decline was reduced by approximately 47% in TG myocytes, indicating decreased SR Ca2+ uptake function. Caffeine-induced Ca2+ transients indicated unaltered SR Ca2+ content and Na+/Ca2+ exchange function. Phosphorylation assays revealed an approximately 30% decrease in the phosphorylation of ryanodine receptor Ser2809. Iso stimulation increased the phosphorylation of both phospholamban Ser16 and the ryanodine receptor Ser2809 but not phospholamban Thr17 in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca2+ handling in TG hearts.     

Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo

Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-epsilon, may slow the rates of cardiac contraction and relaxation (+dP/dt and -dP/dt). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser(43) and Ser(45) and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater -dP/dt than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/dt increased by 33% and -dP/dt increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.     

The transgenic expression of highly inhibitory monomeric forms of phospholamban in mouse heart impairs cardiac contractility

Transgenic mice were generated with cardiac-specific overexpression of the monomeric, dominant-acting, superinhibitory L37A and I40A mutant forms of phospholamban (PLN), and their phenotypes were compared with wild-type (wt) mice or 2-fold overexpressors of wt PLN (wtOE). The level of PLN monomer in cardiac microsomes was increased 11-13-fold, and the apparent affinity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase for Ca(2+) was decreased from pCa 6.22 in wt or 6.12 in wtOE to 5.81 in L37A and 5.72 in I40A. Basal physiological parameters, measured in isolated myocytes, indicated a significant reduction in the rates of shortening (+dL/dt) and relengthening (-dL/dt). Hemodynamic measurements indicated that peak systolic pressure was unaffected but that pressure changes (+dP/dt and -dP/dt) were lowered significantly in both mutant lines, and relaxation time (tau) was also lengthened significantly. Echocardiography for both mutants showed depressed systolic function and an increase in left ventricular mass of over 1.4-fold. Significant decreases in left ventricular shortening fraction and velocity of circumferential shortening and increases in ejection time were corrected by isoproterenol. The use of antibodies specific against Ser(16)- and Thr(17)-PLN peptides showed that phosphorylation of both pentameric and monomeric PLN were increased between 1.2- and 2.4-fold in both the L37A and I40A lines but not in the wtOE line. These observations show that overexpression of superinhibitory mutant forms of PLN causes depression of contractile parameters with induction of cardiac hypertrophy, as assessed with echocardiography.     

PKC-{epsilon}-dependent survival signals in diabetic hearts

Diabetes mellitus is complicated by the development of a primary cardiomyopathy, which contributes to the excess morbidity and mortality of this disorder. The protein kinase C (PKC) family of isozymes plays a key role in the cardiac phenotype expressed during postnatal development and in response to pathological stimuli. Hyperglycemia is an activating signal for cardiac PKC isozymes that modulate a myriad of cell events including cell death and survival. The epsilon-isozyme of the PKC family transmits a powerful survival signal in cardiac muscle cells. Accordingly, to test the hypothesis that endogenous activation of cardiac PKC-epsilon will protect against hyperglycemic cell injury and left ventricular dysfunction, diabetes mellitus was induced using streptozotocin in genetically engineered mice with cardiac-specific expression of the PKC-epsilon translocation activator [psiepsilon-receptors for activated C kinase (psiepsilon-RACK)]. The results demonstrate a striking PKC-epsilon cardioprotective phenotype in diabetic psiepsilon-RACK (epsilon-agonist) mice that is characterized by inhibition of the hyperglycemia apoptosis signal, attenuation of hyperglycemia-mediated oxidative stress, and preservation of parameters of left ventricular pump function. Hearts of diabetic epsilon-agonist mice exhibited selective trafficking of PKC-epsilon to membrane and mitochondrial compartments, phosphorylation/inactivation of the mitochondrial Bad protein, and inhibition of cytochrome c release. We conclude that activation of endogenous PKC-epsilon in hearts of diabetic epsilon-agonist mice promotes the survival phenotype, attenuates markers of oxidative stress, and inhibits the negative inotropic properties of chronic hyperglycemia.     

Ataxia telangiectasia mutated kinase plays a protective role in ��-adrenergic receptor-stimulated cardiac myocyte apoptosis and myocardial remodeling

β-Adrenergic receptor (β-AR) stimulation induces cardiac myocyte apoptosis and plays an important role in myocardial remodeling. Here we investigated expression of various apoptosis-related genes affected by β-AR stimulation, and examined first time the role of ataxia telangiectasia mutated kinase (ATM) in cardiac myocyte apoptosis and myocardial remodeling following β-AR stimulation. cDNA array analysis of 96 apoptosis-related genes indicated that β-AR stimulation increases expression of ATM in the heart. In vitro, RT-PCR confirmed increased ATM expression in adult cardiac myocytes in response to β-AR stimulation. Analysis of left ventricular structural and functional remodeling of the heart in wild-type (WT) and ATM heterozygous knockout mice (hKO) 28 days after ISO-infusion showed increased heart weight to body weight ratio in both groups. M-mode echocardiography showed increased percent fractional shortening (%FS) and ejection fraction (EF%) in both groups 28 days post ISO-infusion. Interestingly, the increase in %FS and EF% was significantly lower in the hKO-ISO group. Cardiac fibrosis and myocyte apoptosis were higher in hKO mice at baseline and ISO-infusion increased fibrosis and apoptosis to a greater extent in hKO-ISO hearts. ISO-infusion increased phosphorylation of p53 (Serine-15) and expression of p53 and Bax to a similar extent in both groups. hKO-Sham and hKO-ISO hearts exhibited reduced intact β1 integrin levels. MMP-2 protein levels were significantly higher, while TIMP-2 protein levels were lower in hKO-ISO hearts. MMP-9 protein levels were increased in WT-ISO, not in hKO hearts. In conclusion, ATM plays a protective role in cardiac remodeling in response to β-AR stimulation.     

Disruption of Rac1 signaling reduces ischemia-reperfusion injury in the diabetic heart by inhibiting calpain

Diabetes increases myocardial ischemia/reperfusion (I/R) injury. However, the underlying mechanisms remain incompletely understood. This study investigated the role of Rac1 signaling and calpain in exacerbated I/R injury in diabetic hearts. Mice with cardiac-specific deletion of Rac1 (Rac1-ko) and transgenic mice with cardiac-specific superoxide dismutase-2 (SOD2) or calpastatin overexpression were rendered diabetic with streptozotocin. Isolated perfused hearts were subjected to global I/R. After I/R, Rac1 activity was significantly enhanced in diabetic compared with nondiabetic hearts. Diabetic hearts displayed more severe I/R injury than nondiabetic hearts, as evidenced by more lactate dehydrogenase release and apoptosis and decreased cardiac function. These adverse impacts of diabetes were abrogated in Rac1-ko hearts or by perfusion with the Rac1 inhibitor NSC23766. In an in vivo I/R mouse model, infarct size was much smaller in diabetic Rac1-ko compared with wild-type mice. Inhibition of Rac1 signaling prevented NADPH oxidase activation, reactive oxygen species production, and protein carbonyl accumulation, leading to inhibition of calpain activation. Furthermore, SOD2 or calpastatin overexpression significantly reduced I/R injury in diabetic hearts and improved cardiac function after I/R. In summary, Rac1 activation increases I/R injury in diabetic hearts and the role of Rac1 signaling is mediated, at least in part, through calpain activation.     

Targeted inhibition of Ca2+/calmodulin-dependent protein kinase II in cardiac longitudinal sarcoplasmic reticulum results in decreased phospholamban phosphorylation at threonine 17

To investigate the role of Ca2+/calmodulin-dependent kinase II in cardiac sarcoplasmic reticulum function, transgenic mice were designed and generated to target the expression of a Ca2+/calmodulin-dependent kinase II inhibitory peptide in cardiac longitudinal sarcoplasmic reticulum using a truncated phospholamban transmembrane domain. The expressed inhibitory peptide was highly concentrated in cardiac sarcoplasmic reticulum. This resulted in a 59.7 and 73.6% decrease in phospholamban phosphorylation at threonine 17 under basal and beta-adrenergic stimulated conditions without changing phospholamban phosphorylation at serine 16. Sarcoplasmic reticulum Ca2+ uptake assays showed that the Vmax was decreased by approximately 30% although the apparent affinity for Ca2+ was unchanged in heterozygous hearts. The in vivo measurement of cardiac function showed no significant reductions in positive and negative dP/dt, but a moderate 18% decrease in dP/dt40, indicative of isovolumic contractility, and a 26.1% increase in the time constant of relaxation (tau) under basal conditions. The changes in these parameters indicate a moderate cardiac dysfunction in transgenic mice. Although the 3 and 4-month-old transgenic mice displayed no overt signs of cardiac disease, when stressed by gestation and parturition, the 7-month-old female mice develop dilated heart failure, suggesting the important role of Ca2+/calmodulin-dependent kinase II pathway in the development of cardiac disease.     

Disruption of protein kinase A interaction with A-kinase-anchoring proteins in the heart in vivo: effects on cardiac contractility, protein kinase A phosphorylation, and troponin I proteolysis

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.     

Oncostatin M (OSM) protects against cardiac ischaemia/reperfusion injury in diabetic mice by regulating apoptosis,mitochondrial biogenesis and insulin sensitivity

Oncostatin M (OSM) exhibits many unique biological activities by activating Oβ receptor. However, its role in myocardial I/R injury in diabetic mice remains unknown. The involvement of OSM was assessed in diabetic mice which underwent myocardial I/R injury by OSM treatment or genetic deficiency of OSM receptor Oβ. Its mechanism on cardiomyocyte apoptosis, mitochondrial biogenesis and insulin sensitivity were further studied. OSM alleviated cardiac I/R injury by inhibiting cardiomyocyte apoptosis through inhibition of inositol pyrophosphate 7 (IP7) production, thus activating PI3K/Akt/BAD pathway, decreasing Bax expression while up‐regulating Bcl‐2 expression and decreasing the ratio of Bax to Bcl‐2 in db/db mice. OSM enhanced mitochondrial biogenesis and mitochondrial function in db/db mice subjected to cardiac I/R injury. On the contrary, OSM receptor Oβ knockout exacerbated cardiac I/R injury, increased IP7 production, enhanced cardiomyocyte apoptosis, impaired mitochondrial biogenesis, glucose homoeostasis and insulin sensitivity in cardiac I/R injured diabetic mice. Inhibition of IP7 production by TNP (IP6K inhibitor) exerted similar effects of OSM. The mechanism of OSM on cardiac I/R injury in diabetic mice is partly associated with IP7/Akt and adenine mononucleotide protein kinase/PGC‐1α pathway. OSM protects against cardiac I/R Injury by regulating apoptosis, insulin sensitivity and mitochondrial biogenesis in diabetic mice through inhibition of IP7 production.     

Diabetic alterations in cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban protein expression.

Diabetic cardiomyopathy has been suggested to be caused by abnormal intracellular Ca2+ homeostasis in the myocardium, which is partly due to a defect in calcium transport by the cardiac sarcoplasmic reticulum (SR). In the present study, the underlying mechanism for this functional derangement was investigated with respect to SR Ca2+-ATPase and phospholamban (the inhibitor of SR Ca2+-ATPase). The maximal Ca2+ uptake and the affinity of Ca2+-ATPase for Ca2+ were decreased, and exogenous phosphorylation level of phospholamban was higher in streptozotocin-induced diabetic rat SR. Levels of both mRNA and protein of phospholamban were significantly increased in the diabetic hearts, whereas those of SR Ca2+-ATPase were significantly decreased. Consequently, the relative phospholamban/Ca2+-ATPase ratio was 1.88 in the diabetic hearts, and these changes were correlated with changes in the rates of SR Ca2+ uptake. However, phosphatase pretreatment of phospholamban for dephosphorylation of the sites phosphorylated in vivo did not change the levels of subsequent phospholamban phosphorylation in either control or diabetic rat hearts. The above data indicated that the increased phospholamban phosphorylation was not due to autonomic dysfunction but possibly due to increased phospholamban expression. These findings suggest that reduction of the SR Ca2+-ATPase level would contribute to decreased rates of SR Ca2+ uptake and that this function is further impaired by the enhanced inhibition by phospholamban due to its increased expression in the diabetic heart.     

Maximal inhibition of SERCA2 Ca(2+) affinity by phospholamban in transgenic hearts overexpressing a non-phosphorylatable form of phospholamban

Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR) which regulates the apparent Ca(2+) affinity of the SR Ca(2+)-ATPase (SERCA2). To determine the levels of phospholamban which are associated with maximal inhibition of SERCA2, several lines of transgenic mice were generated which expressed increasing levels of a non-phosphorylatable form of phospholamban (S16A,T17A) specifically in the heart. This mutant form of phospholamban was chosen to prevent phosphorylation as a compensatory mechanism in vivo. Quantitative immunoblotting revealed increased phospholamban protein levels of 1.8-, 2.6-, 3.7-, and 4.7-fold in transgenic hearts compared with wild types. There were no changes in the expression levels of SERCA2, calsequestrin, calreticulin, and ryanodine receptor. Assessment of SR Ca(2+) uptake in hearts of transgenic mice indicated increases in the inhibition of the affinity of SERCA2 for Ca(2+) with increased phospholamban expression. Maximal inhibition was obtained at phospholamban expression levels of 2.6-fold or higher. Transgenic hearts with functional saturation in phospholamban:SERCA2 (>/=2.6:1) exhibited increases in beta-myosin heavy chain expression, associated with cardiac hypertrophy. These findings demonstrate that overexpression of a non-phosphorylatable form of phospholamban in transgenic mouse hearts resulted in saturation of the functional phospholamban:SERCA2 ratio at 2.6:1 and suggest that approximately 40% of the SR Ca(2+) pumps are functionally regulated by phospholamban in vivo.     

Myosin regulatory light chain phosphorylation attenuates cardiac hypertrophy

Hyperphosphorylation of myosin regulatory light chain (RLC) in cardiac muscle is proposed to cause compensatory hypertrophy. We therefore investigated potential mechanisms in genetically modified mice. Transgenic (TG) mice were generated to overexpress Ca2+/calmodulin-dependent myosin light chain kinase specifically in cardiomyocytes. Phosphorylation of sarcomeric cardiac RLC and cytoplasmic nonmuscle RLC increased markedly in hearts from TG mice compared with hearts from wild-type (WT) mice. Quantitative measures of RLC phosphorylation revealed no spatial gradients. No significant hypertrophy or structural abnormalities were observed up to 6 months of age in hearts of TG mice compared with WT animals. Hearts and cardiomyocytes from WT animals subjected to voluntary running exercise and isoproterenol treatment showed hypertrophic cardiac responses, but the responses for TG mice were attenuated. Additional biochemical measurements indicated that overexpression of the Ca2+/calmodulin-binding kinase did not perturb other Ca2+/calmodulin-dependent processes involving Ca2+/calmodulin-dependent protein kinase II or the protein phosphatase calcineurin. Thus, increased myosin RLC phosphorylation per se does not cause cardiac hypertrophy and probably inhibits physiological and pathophysiological hypertrophy by contributing to enhanced contractile performance and efficiency.     

Rescue of contractile parameters and myocyte hypertrophy in calsequestrin overexpressing myocardium by phospholamban ablation

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.     

Diminished Autophagy Limits Cardiac Injury in Mouse Models of Type 1 Diabetes

Cardiac autophagy is inhibited in type 1 diabetes. However, it remains unknown if the reduced autophagy contributes to the pathogenesis of diabetic cardiomyopathy. We addressed this question using mouse models with gain- and loss-of-autophagy. Autophagic flux was inhibited in diabetic hearts when measured at multiple time points after diabetes induction by streptozotocin as assessed by protein levels of microtubule-associated protein light chain 3 form 2 (LC3-II) or GFP-LC3 puncta in the absence and presence of the lysosome inhibitor bafilomycin A1. Autophagy in diabetic hearts was further reduced in beclin 1- or Atg16-deficient mice but was restored partially or completely by overexpression of beclin 1 to different levels. Surprisingly, diabetes-induced cardiac damage was substantially attenuated in beclin 1- and Atg16-deficient mice as shown by improved cardiac function as well as reduced levels of oxidative stress, interstitial fibrosis, and myocyte apoptosis. In contrast, diabetic cardiac damage was dose-dependently exacerbated by beclin 1 overexpression. The cardioprotective effects of autophagy deficiency were reproduced in OVE26 diabetic mice. These effects were associated with partially restored mitophagy and increased expression and mitochondrial localization of Rab9, an essential regulator of a non-canonical alternative autophagic pathway. Together, these findings demonstrate that the diminished autophagy is an adaptive response that limits cardiac dysfunction in type 1 diabetes, presumably through up-regulation of alternative autophagy and mitophagy.     

Cardiac-specific overexpression of a superinhibitory pentameric phospholamban mutant enhances inhibition of cardiac function in vivo

Phospholamban is a regulator of the Ca(2+) affinity of the cardiac sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) and of cardiac contractility. In vitro expression studies have shown that several mutant phospholamban monomers are superinhibitory, suggesting that monomeric phospholamban is the active species. However, a phospholamban Asn(27) --> Ala (N27A) mutant, which maintained a normal pentamer to monomer ratio, was shown to act as a superinhibitor of SERCA2a Ca(2+) affinity. To determine whether the pentameric N27A mutant is superinhibitory in vivo, transgenic mice with cardiac-specific overexpression of mutant phospholamban were generated. Quantitative immunoblotting revealed a 61 +/- 6% increase in total phospholamban in mutant hearts, with 90% of the overexpressed protein being pentameric. The EC(50) value for Ca(2+) dependence of Ca(2+) uptake was 0.69 +/- 0.07 microM in mutant hearts, compared with 0.29 +/- 0.02 microM in wild-type hearts or 0. 43 +/- 0.03 microM in hearts overexpressing wild-type PLB by 2-fold. Myocytes from phospholamban N27A mutant hearts also exhibited more depressed contractile parameters than wild-type phospholamban overexpressing cells. The shortening fraction was 52%, rates of shortening and relengthening were 46% and 38% respectively, and time for 80% decay of the Ca(2+) signal was 146%, compared with wild-types (100%). Langendorff-perfused mutant hearts also demonstrated depressed contractile parameters. Furthermore, in vivo echocardiography showed a depression in the ratio of early to late diastolic transmitral velocity and a 79% prolongation of the isovolumic relaxation time. Isoproterenol stimulation did not fully relieve the depressed contractile parameters at the cellular, organ, and intact animal levels. Thus, pentameric phospholamban N27A mutant can act as a superinhibitor of the affinity of SERCA2a for Ca(2+) and of cardiac contractility in vivo.     

Glycogen synthase kinase-3alpha reduces cardiac growth and pressure overload-induced cardiac hypertrophy by inhibition of extracellular signal-regulated kinases

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase having multiple functions and consisting of two isoforms, GSK-3alpha and GSK-3beta. Pressure overload increases expression of GSK-3alpha but not GSK-3beta. Despite our wealth of knowledge about GSK-3beta, the function of GSK-3alpha in the heart is not well understood. To address this issue, we made cardiac-specific GSK-3alpha transgenic mice (Tg). Left ventricular weight and cardiac myocyte size were significantly smaller in Tg than in non-Tg (NTg) mice, indicating that GSK-3alpha inhibits cardiac growth. After 4 weeks of aortic banding (transverse aortic constriction (TAC)), increases in left ventricular weight and myocyte size were significantly smaller in Tg than in NTg, indicating that GSK-3alpha inhibits cardiac hypertrophy. More severe cardiac dysfunction developed in Tg after TAC. Increases in fibrosis and apoptosis were greater in Tg than in NTg after TAC. Among signaling molecules screened, ERK phosphorylation was decreased in Tg. Adenovirus-mediated overexpression of GSK-3alpha, but not GSK-3beta, inhibited ERK in cultured cardiac myocytes. Knockdown of GSK-3alpha increased ERK phosphorylation, an effect that was inhibited by PD98059, rottlerin, and protein kinase Cepsilon (PKCepsilon) inhibitor peptide, suggesting that GSK-3alpha inhibits ERK through PKC-MEK-dependent mechanisms. Knockdown of GSK-3alpha increased protein content and reduced apoptosis, effects that were abolished by PD98059, indicating that inhibition of ERK plays a major role in the modulation of cardiac growth and apoptosis by GSK-3alpha. In conclusion, up-regulation of GSK-3alpha inhibits cardiac growth and pressure overload-induced cardiac hypertrophy but increases fibrosis and apoptosis in the heart. The anti-hypertrophic and pro-apoptotic effect of GSK-3alpha is mediated through inhibition of ERK.     

Lack of osteopontin improves cardiac function in streptozotocin-induced diabetic mice

The purpose of this study was to investigate the role of osteopontin (OPN) in diabetic hearts. Diabetes was induced in wild-type (WT) and OPN knockout (KO) mice by using streptozotocin (150 mg/kg) injection. Left ventricular (LV) structural and functional remodeling was studied 30 and 60 days after induction of diabetes. Induction of diabetes increased OPN expression in cardiac myocytes. Heart weight-to-body weight ratio was increased in both diabetic (D) groups. Lung wet weight-to-dry weight ratio was increased only in the WT-D group. Peak left ventricular (LV) developed pressures measured using Langendorff perfusion analyses were reduced to a greater extent in WT-D versus KO-D group. LV end-diastolic pressure-volume curve exhibited a significant leftward shift in WT-D but not in KO-D group. LV end-diastolic diameter, percent fractional shortening, and the ratio of peak velocity of early and late filling (E/A wave) were significantly reduced in WT-D mice as analyzed by echocardiography. The increase in cardiac myocyte apoptosis and fibrosis was significantly higher in the WT-D group. Expression of atrial natriuretic peptide and transforming growth factor-beta1 was significantly increased in the WT-D group. Induction of diabetes increased protein kinase C (PKC) phosphorylation in both groups. However, phosphorylation of PKC-betaII was significantly higher in the WT-D group, whereas phosphorylation of PKC-zeta was significantly higher in the KO-D group. Levels of peroxisome proliferator-activated receptor-gamma were significantly decreased in the WT-D group but not in the KO-D group. Thus increased expression of OPN may play a deleterious role during streptozotocin-induced diabetic cardiomyopathy with effects on cardiac fibrosis, hypertrophy, and myocyte apoptosis.     

PKCepsilon increases phosphorylation of the cardiac myosin binding protein C at serine 302 both in vitro and in vivo

Cardiac myosin binding protein C (cMyBPC) phosphorylation is essential for normal cardiac function. Although PKC was reported to phosphorylate cMyBPC in vitro, the relevant PKC isoforms and functions of PKC-mediated cMyBPC phosphorylation are unknown. We recently reported that a transgenic mouse model with cardiac-specific overexpression of PKCepsilon (PKCepsilon TG) displayed enhanced sarcomeric protein phosphorylation and dilated cardiomyopathy. In the present study, we have investigated cMyBPC phosphorylation in PKCepsilon TG mice. Western blotting and two-dimensional gel electrophoresis demonstrated a significant increase in cMyBPC serine (Ser) phosphorylation in 12-month-old TG mice compared to wild type (WT). In vitro PKCepsilon treatment of myofibrils increased the level of cMyBPC Ser phosphorylation in WT mice to that in TG mice, whereas treatment of TG myofibrils with PKCepsilon showed only a minimal increase in cMyBPC Ser phosphorylation. Three peptide motifs of cMyBPC were identified as the potential PKCepsilon consensus sites including a 100% matched motif at Ser302 and two nearly matched motifs at Ser811 and Ser1203. We treated synthetic peptides corresponding to the sequences of these three motifs with PKCepsilon and determined phosphorylation by mass spectrometry and ELISA assay. PKCepsilon induced phosphorylation at the Ser302 site but not at the Ser811 or Ser1203 sites. A S302A point mutation in the Ser302 peptide abolished the PKCepsilon-dependent phosphorylation. Taken together, our data show that the Ser302 on mouse cMyBPC is a likely PKCepsilon phosphorylation site both in vivo and in vitro and may contribute to the dilated cardiomyopathy associated with increased PKCepsilon activity.