Cardiac myosin binding protein-C phosphorylation in a {beta}-myosin heavy chain background.

BACKGROUND: Cardiac myosin binding protein-C (cMyBP-C) phosphorylation modulates cardiac contractility. When expressed in cMyBP-C-null (cMyBP-C((t/t))) hearts, a cMyBP-C phosphomimetic (cMyBP-C(AllP+)) rescued cardiac dysfunction and protected the hearts from ischemia/reperfusion injury. However, cMyBP-C function may be dependent on the myosin isoform type. Because these replacements were performed in the mouse heart, which contains predominantly alpha-myosin heavy chain (alpha-MyHC), the applicability of the data to humans, whose cardiomyocytes contain predominantly beta-MyHC, is unclear. We determined the effect(s) of cMyBP-C phosphorylation in a beta-MyHC transgenic mouse heart in which >80% of the alpha-MyHC was replaced by beta-MyHC, which is the predominant myosin isoform in human cardiac muscle. METHODS AND RESULTS: To determine the effects of cMyBP-C phosphorylation in a beta-MyHC background, transgenic mice expressing normal cMyBP-C (cMyBP-C(WT)), nonphosphorylatable cMyBP-C (cMyBP-C(AllP)(-)), or cMyBP-C(AllP+) were bred into the beta-MyHC background (beta). These mice were then crossed into the cMyBP-C((t/t)) background to ensure the absence of endogenous cMyBP-C. cMyBP-C((t/t)/beta) and cMyBP-C(AllP)(-)(:(t/t)/beta) mice died prematurely because of heart failure, confirming that cMyBP-C phosphorylation is essential in the beta-MyHC background. cMyBP-C(AllP+:(t/t)/beta) and cMyBP-C(WT:(t/t)/beta) hearts showed no morbidity and mortality, and cMyBP-C(AllP+:(t/t)/beta) hearts were significantly cardioprotected from ischemia/reperfusion injury. CONCLUSIONS: cMyBP-C phosphorylation is necessary for basal myocardial function in the beta-MyHC background and can preserve function after ischemia/reperfusion injury. Our studies justify exploration of cMyBP-C phosphorylation as a therapeutic target in the human heart.

Cardiomyocyte expression of a polyglutamine preamyloid oligomer causes heart failure.

BACKGROUND: To determine whether soluble preamyloid oligomers (PAOs) are toxic when expressed internally in the cardiomyocyte, we tested the hypothesis that cardiomyocyte-restricted expression and accumulation of a known PAO is cytotoxic and sufficient to cause heart failure. METHODS AND RESULTS: Intracellular PAOs, the entities believed to cause toxicity in many neurodegenerative diseases, have been observed in cardiomyocytes derived from mouse and human heart failure samples. Long (>50) polyglutamine (PQ) repeats form PAOs and cause neurotoxicity in Huntington disease and other neurodegenerative diseases, whereas shorter PQ peptides are benign. We created transgenic mice in which cardiomyocyte-autonomous expression of an 83 residue-long PQ repeat (PQ83) or a non-amyloid-forming peptide of 19 PQ repeats (PQ19) as a nonpathological control was expressed. A PQ83 line with relatively low levels of expression was generated, along with a PQ19 line that expressed approximately 9-fold the levels observed in the PQ83 line. Hearts expressing PQ83 exhibited reduced cardiac function and dilation by 5 months, and all mice died by 8 months, whereas PQ19 mice had normal cardiac function, morphology, and life span. PQ83 protein accumulated within aggresomes with PAO-specific staining. The PQ83 hearts showed increased autophagosomal and lysosomal content but also showed markers of necrotic death, including inflammatory cell infiltration and increased sarcolemmal permeability. CONCLUSIONS: The data confirm the hypothesis that expression of an exogenous PAO-forming peptide is toxic to cardiomyocytes and is sufficient to cause cardiomyocyte loss and heart failure in a murine model.

Role of the acidic N' region of cardiac troponin I in regulating myocardial function.

Cardiac troponin I (cTnI) phosphorylation modulates myocardial contractility and relaxation during beta-adrenergic stimulation. cTnI differs from the skeletal isoform in that it has a cardiac specific N' extension of 32 residues (N' extension). The role of the acidic N' region in modulating cardiac contractility has not been fully defined. To test the hypothesis that the acidic N' region of cTnI helps regulate myocardial function, we generated cardiac-specific transgenic mice in which residues 2-11 (cTnI(Delta2-11)) were deleted. The hearts displayed significantly decreased contraction and relaxation under basal and beta-adrenergic stress compared to nontransgenic hearts, with a reduction in maximal Ca(2+)-dependent force and maximal Ca(2+)-activated Mg(2+)-ATPase activity. However, Ca(2+) sensitivity of force development and cTnI-Ser(23/24) phosphorylation were not affected. Chemical shift mapping shows that both cTnI and cTnI(Delta2-11) interact with the N lobe of cardiac troponin C (cTnC) and that phosphorylation at Ser(23/24) weakens these interactions. These observations suggest that residues 2-11 of cTnI, comprising the acidic N' region, do not play a direct role in the calcium-induced transition in the cardiac regulatory or N lobe of cTnC. We hypothesized that phosphorylation at Ser(23/24) induces a large conformational change positioning the conserved acidic N region to compete with actin for the inhibitory region of cTnI. Consistent with this hypothesis, deletion of the conserved acidic N' region results in a decrease in myocardial contractility in the cTnI(Delta2-11) mice demonstrating the importance of acidic N' region in regulating myocardial contractility and mediating the response of the heart to beta-AR stimulation.

Phospholamban overexpression in transgenic rabbits.

There has been considerable interest in pursuing phospholamban as a putative therapeutic target for overcoming depressed calcium handling in human heart failure. Studies predominantly done in mice have shown that phospholamban is a key regulator of sarcoplasmic reticulum calcium cycling and cardiac function. However, mice differ significantly from humans in how they regulate calcium, whereas rabbits better recapitulate human cardiac function and calcium handling. To investigate phospholamban's role in the rabbit heart, transgenic rabbits that overexpressed wild-type phospholamban in the ventricular cardiomyocytes and slow-twitch skeletal muscles were generated. Rabbits expressing high levels of phospholamban were not viable due to severe skeletal muscle wasting, the onset of cardiac pathology and early death. A viable transgenic line exhibited a 30% increase in PLN protein levels in the heart. These animals showed isolated foci of cardiac pathology, but cardiac function as well as the response to beta-adrenergic stimulation were normal. SR-calcium uptake measurements showed that the transgenic hearts had the expected reduced affinity for calcium. The data show that phospholamban-overexpressing transgenic rabbits differ markedly in phenotype from analogous transgenic mice in that rabbits are quite sensitive to alterations in phospholamban levels. Exceeding a relatively narrow window of phospholamban expression results in significant morbidity and early death.

Cardiomyocyte GATA4 functions as a stress-responsive regulator of angiogenesis in the murine heart.

The transcription factor GATA4 is a critical regulator of cardiac gene expression, modulating cardiomyocyte differentiation and adaptive responses of the adult heart. We report what we believe to be a novel function for GATA4 in murine cardiomyocytes as a nodal regulator of cardiac angiogenesis. Conditional overexpression of GATA4 within adult cardiomyocytes increased myocardial capillary and small conducting vessel densities and increased coronary flow reserve and perfusion-dependent cardiac contractility. Coculture of HUVECs with either GATA4-expressing cardiomyocytes or with myocytes expressing a dominant-negative form of GATA4 enhanced or reduced HUVEC tube formation, respectively. Expression of GATA4 in skeletal muscle by adenoviral gene transfer enhanced capillary densities and hindlimb perfusion following femoral artery ablation. Deletion of Gata4 specifically from cardiomyocytes reduced myocardial capillary density and prevented pressure overload-augmented angiogenesis in vivo. GATA4 induced the angiogenic factor VEGF-A, directly binding the Vegf-A promoter and enhancing transcription. GATA4-overexpressing mice showed increased levels of cardiac VEGF-A, while Gata4-deleted mice demonstrated decreased VEGF-A levels. The induction of HUVEC tube formation in GATA4-overexpressing cocultured myocytes was blocked with a VEGF receptor antagonist. Pressure overload-induced dysfunction in Gata4-deleted hearts was partially rescued by adenoviral gene delivery of VEGF and angiopoietin-1. To our knowledge, these results demonstrate [corrected] a previously unrecognized function for GATA4 as a regulator of cardiac angiogenesis through a nonhypoxic, load, and/or disease-responsive mechanism.

Ca2+- and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure.

Loss of cardiac myocytes in heart failure is thought to occur largely through an apoptotic process. Here we show that heart failure can also be precipitated through myocyte necrosis associated with Ca2+ overload. Inducible transgenic mice with enhanced sarcolemmal L-type Ca2+ channel (LTCC) activity showed progressive myocyte necrosis that led to pump dysfunction and premature death, effects that were dramatically enhanced by acute stimulation of beta-adrenergic receptors. Enhanced Ca2+ influx-induced cellular necrosis and cardiomyopathy was prevented with either LTCC blockers or beta-adrenergic receptor antagonists, demonstrating a proximal relationship among beta-adrenergic receptor function, Ca2+ handling, and heart failure progression through necrotic cell loss. Mechanistically, loss of cyclophilin D, a regulator of the mitochondrial permeability transition pore that underpins necrosis, blocked Ca2+ influx-induced necrosis of myocytes, heart failure, and isoproterenol-induced premature death. In contrast, overexpression of the antiapoptotic factor Bcl-2 was ineffective in mitigating heart failure and death associated with excess Ca2+ influx and acute beta-adrenergic receptor stimulation. This paradigm of mitochondrial- and necrosis-dependent heart failure was also observed in other mouse models of disease, which supports the concept that heart failure is a pleiotropic disorder that involves not only apoptosis, but also necrotic loss of myocytes in association with dysregulated Ca2+ handling and beta-adrenergic receptor signaling.

Cardiac myosin binding protein C phosphorylation is cardioprotective.

Cardiac myosin binding protein C (cMyBP-C) has three phosphorylatable serines at its N terminus (Ser-273, Ser-282, and Ser-302), and the residues' phosphorylation states may alter thick filament structure and function. To examine the effects of cMyBP-C phosphorylation, we generated transgenic mice with cardiac-specific expression of a cMyBP-C in which the three phosphorylation sites were mutated to aspartic acid, mimicking constitutive phosphorylation (cMyBP-C(AllP+)). The allele was bred into a cMyBP-C null background (cMyBP-C((t/t))) to ensure the absence of endogenous dephosphorylated cMyBP-C. cMyBP-C(AllP+) was incorporated normally into the cardiac sarcomere and restored normal cardiac function in the null background. However, subtle changes in sarcomere ultrastructure, characterized by increased distances between the thick filaments, indicated that phosphomimetic cMyBP-C affects thick-thin filament relationships, and yeast two-hybrid data and pull-down studies both showed that charged residues in these positions effectively prevented interaction with the myosin heavy chain. Confirming the physiological relevance of these data, the cMyBP-C(AllP+:(t/t)) hearts were resistant to ischemia-reperfusion injury. These data demonstrate that cMyBP-C phosphorylation functions in maintaining thick filament spacing and structure and can help protect the myocardium from ischemic injury.

Pharmacological- and gene therapy-based inhibition of protein kinase Calpha/beta enhances cardiac contractility and attenuates heart failure.

BACKGROUND: The conventional protein kinase C (PKC) isoform alpha functions as a proximal regulator of Ca2+ handling in cardiac myocytes. Deletion of PKCalpha in the mouse results in augmented sarcoplasmic reticulum Ca2+ loading, enhanced Ca2+ transients, and augmented contractility, whereas overexpression of PKCalpha in the heart blunts contractility. Mechanistically, PKCalpha directly regulates Ca2+ handling by altering the phosphorylation status of inhibitor-1, which in turn suppresses protein phosphatase-1 activity, thus modulating phospholamban activity and secondarily, the sarcoplasmic reticulum Ca2+ ATPase. METHODS AND RESULTS: In the present study, we show that short-term inhibition of the conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 significantly augmented cardiac contractility in vivo or in an isolated work-performing heart preparation in wild-type mice but not in PKCalpha-deficient mice. Ro-32-0432 also increased cardiac contractility in 2 different models of heart failure in vivo. Short-term or long-term treatment with Ro-31-8220 in a mouse model of heart failure due to deletion of the muscle lim protein gene significantly augmented cardiac contractility and restored pump function. Moreover, adenovirus-mediated gene therapy with a dominant-negative PKCalpha cDNA rescued heart failure in a rat model of postinfarction cardiomyopathy. PKCalpha was also determined to be the dominant conventional PKC isoform expressed in the adult human heart, providing potential relevance of these findings to human pathophysiology. CONCLUSIONS: Pharmacological inhibition of PKCalpha, or the conventional isoforms in general, may serve as a novel therapeutic strategy for enhancing cardiac contractility in certain stages of heart failure.

Changes in end-to-end interactions of tropomyosin affect mouse cardiac muscle dynamics.

The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size and regulating the affinity of TM for actin. We hypothesized that altering the alpha-TM carboxy terminal overlap end to the beta-TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we generated transgenic (TG) mouse lines that express a mutant form of alpha-TM in which the first 275 residues are from alpha-TM and the last nine amino acids are from beta-TM (alpha-TM9aaDeltabeta). Molecular analyses show that endogenous alpha-TM mRNA and protein are nearly completely replaced with alpha-TM9aaDeltabeta. Working heart preparations data show that the rates of contraction and relaxation are reduced in alpha-TM9aaDeltabeta hearts. Left ventricular pressure and time to peak pressure are also reduced (-12% and -13%, respectively). The ratio of maximum to minimum first derivatives of change in left ventricular systolic pressure with respect to time (ratio of +dP/dt to -dP/dt, respectively) is increased, but tau is not changed significantly. Force-intracellular calcium concentration ([Ca2+]i) measurements from intact papillary fibers demonstrate that alpha-TM9aaDeltabeta TG fibers produce less force per given [Ca2+]i compared with nontransgenic fibers. Taken together, the data demonstrate that the rate of contraction is primarily affected in TM TG hearts. Protein docking studies show that in the mutant molecule, the overall carbon backbone is perturbed about 1.5 A, indicating that end-to-end interactions are altered. These results demonstrate that the localized flexibility present in the coiled-coil structures of TM isoforms is different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and with differentially modulating the myofilament activation processes.

GDF15/MIC-1 functions as a protective and antihypertrophic factor released from the myocardium in association with SMAD protein activation.

Here we identified growth-differentiation factor 15 (GDF15) (also known as MIC-1), a secreted member of the transforming growth factor (TGF)-beta superfamily, as a novel antihypertrophic regulatory factor in the heart. GDF15 is not expressed in the normal adult heart but is induced in response to conditions that promote hypertrophy and dilated cardiomyopathy. To elucidate the function of GDF15 in the heart, we generated transgenic mice with cardiac-specific overexpression. GDF15 transgenic mice were normal but were partially resistant to pressure overload-induced hypertrophy. Expression of GDF15 in neonatal cardiomyocyte cultures by adenoviral-mediated gene transfer antagonized agonist-induced hypertrophy in vitro. Transient expression of GDF15 outside the heart by intravenous adenoviral delivery, or by direct injection of recombinant GDF15 protein, attenuated ventricular dilation and heart failure in muscle lim protein gene-targeted mice through an endocrine effect. Conversely, examination of Gdf15 gene-targeted mice showed enhanced cardiac hypertrophic growth following pressure overload stimulation. Gdf15 gene-targeted mice also demonstrated a pronounced loss in ventricular performance following only 2 weeks of pressure overload stimulation, whereas wild-type controls maintained function. Mechanistically, GDF15 stimulation promoted activation of SMAD2/3 in cultured neonatal cardiomyocytes. Overexpression of SMAD2 attenuated cardiomyocyte hypertrophy similar to GDF15 treatment, whereas overexpression of the inhibitory SMAD proteins, SMAD6/7, reversed the antihypertrophic effects of GDF15. These results identify GDF15 as a novel autocrine/endocrine factor that antagonizes the hypertrophic response and loss of ventricular performance, possibly through a mechanism involving SMAD proteins.

Cardiac myosin-binding protein-C phosphorylation and cardiac function.

The role of cardiac myosin binding protein-C (cMyBP-C) phosphorylation in cardiac physiology or pathophysiology is unclear. To investigate the status of cMyBP-C phosphorylation in vivo, we determined its phosphorylation state in stressed and unstressed mouse hearts. cMyBP-C phosphorylation is significantly decreased during the development of heart failure or pathologic hypertrophy. We then generated transgenic (TG) mice in which the phosphorylation sites of cMyBP-C were changed to nonphosphorylatable alanines (MyBP-C(AllP-)). A TG line showing &40% replacement with MyBP-C(AllP-) showed no changes in morbidity or mortality but displayed depressed cardiac contractility, altered sarcomeric structure and upregulation of transcripts associated with a hypertrophic response. To explore the effect of complete replacement of endogenous cMyBP-C with MyBP-C(AllP-), the mice were bred into the MyBP-C(t/t) background, in which less than 10% of normal levels of a truncated MyBP-C are present. Although MyBP-C(AllP-) was incorporated into the sarcomere and expressed at normal levels, the mutant protein could not rescue the MyBP-C(t/t) phenotype. The mice developed significant cardiac hypertrophy with myofibrillar disarray and fibrosis, similar to what was observed in the MyBP-C(t/t) animals. In contrast, when the MyBP-C(t/t) mice were bred to a TG line expressing normal MyBP-C (MyBP-CWT), the MyBP-C(t/t) phenotype was rescued. These data suggest that cMyBP-C phosphorylation is essential for normal cardiac function.

Reversal of amyloid-induced heart disease in desmin-related cardiomyopathy.

Amyloid oligomers, similar to the toxic entities found in Alzheimer's disease patients and in other amyloid-based diseases, are present in cardiomyocytes derived from human heart-failure patients and in animal models of desmin-related cardiomyopathy (DRM). The R120G mutation in alpha-B-crystallin (CryAB) causes DRM and is characterized by aggresomes containing CryAB(R120G) and amyloid oligomer. In this study, we show that aggresome levels do not correlate with disease. Blocking aggresome formation results in increased levels of toxic amyloid oligomer and decreased cardiomyocyte viability. We confirmed the primary toxicity of intrasarcoplasmic amyloid accumulation in the cardiomyocytes by ectopic expression of the amyloidogenic peptide PQ81, which consists of multiple repeats of a polyglutamine tract. We then addressed the issue of disease reversibility by placing CryAB(R120G) under inducible cardiomyocyte-specific expression in transgenic mice. The mice developed aggresomes and contained high concentrations of amyloid oligomer in the heart, resulting in cardiac disease. Cessation of CryAB(R120G) expression in symptomatic mice improved cardiac function and rescued all of the animals from premature death. Rescue was accompanied by significant decreases in amyloid oligomer without a significant reduction in aggresomes. Blocking cardiac amyloid oligomer formation, even after cardiac dysfunction presents, may be a therapeutic strategy in DRM as well as in other types of cardiac disease in which significant amyloid accumulation occurs.

Genetic inhibition or activation of JNK1/2 protects the myocardium from ischemia-reperfusion-induced cell death in vivo.

The c-Jun NH2-terminal kinase (JNK) branch of the mitogen-activated protein kinase signaling cascade has been implicated in the regulation of apoptosis in a variety of mammalian cell types. In the heart, disagreement persists concerning the role that JNKs may play in regulating apoptosis, since both pro- and antiapoptotic regulatory functions have been reported in cultured cardiomyocytes. Here we report the first analysis of cardiomyocyte cell death due to JNK inhibition or activation in vivo using genetically modified mice. Three separate mouse models with selective JNK inhibition were assessed for ventricular damage and apoptosis levels following ischemia-reperfusion injury. jnk1-/-, jnk2-/-, and transgenic mice expressing dominant negative JNK1/2 within the heart were each shown to have less JNK activity in the heart and less injury and cellular apoptosis in vivo following ischemia-reperfusion injury. To potentially address the reciprocal gain-of-function phenotype associated with sustained JNK activation, transgenic mice were generated that express MKK7 in the heart. These transgenic mice displayed elevated cardiac c-Jun kinase activity but, ironically, were also significantly protected from ischemia-reperfusion. Mechanistically, JNK-inhibited mice showed increased phosphorylation of the proapoptotic factor Bad at position 112, whereas MKK7 transgenic mice showed decreased phosphorylation of this site. Collectively, these results underscore the complexity associated with JNK signaling in regulating apoptosis, such that sustained inhibition or activation both elicit cellular protection in vivo, although probably through different mechanisms.

Transgenic rabbit model for human troponin I-based hypertrophic cardiomyopathy.

BACKGROUND: Transgenic and gene-targeted models have focused on the mouse. Fundamental differences between the mouse and human exist in Ca2+ handling during contraction/relaxation and in alterations in Ca2+ flux during heart failure, with the rabbit more accurately reflecting the human system. METHODS AND RESULTS: Cardiac troponin I (cTnI) mutations can cause familial hypertrophic cardiomyopathy. An inhibitory domain mutation, arginine146-->glycine (cTnI(146Gly)), was modeled with the use of transgenic expression in the rabbit ventricle. cTnI(146Gly) levels >40% of total cTnI were perinatally lethal, whereas replacement levels of 15% to 25% were well tolerated. cTnI(146Gly) expression led to a leftward shift in the force-pCa2+ curves with cardiomyocyte disarray, fibrosis, and altered connexin43 organization. In isolated cTnI(146Gly) myocytes, twitch relaxation amplitudes were smaller than in normal cells, but [Ca]i transients and sarcoplasmic reticulum Ca2+ load were not different. Detrended fluctuation analysis of the QT(max) intervals was used to evaluate the cardiac repolarization phase and showed a significantly higher scaling exponent in the transgenic animals. CONCLUSIONS: Expression of modest amounts of cTnI(146Gly) led to subtle defects without severely affecting cardiac function. Aberrant connexin organization, subtle morphological deficits, and an altered fractal pattern of the repolarization phase of transgenic rabbits, in the absence of entropy or other ECG abnormalities, may indicate an early developing pathology before the onset of more obvious repolarization abnormalities or major alterations in cardiac mechanics.

Forced expression of alpha-myosin heavy chain in the rabbit ventricle results in cardioprotection under cardiomyopathic conditions.

BACKGROUND: The biochemical differences between the 2 mammalian cardiac myosin heavy chains (MHCs), alpha-MHC and beta-MHC, are well described, but the physiological consequences of basal isoform expression and isoform shifts in response to altered cardiac load are not clearly understood. Mature human ventricle contains primarily the beta-MHC isoform. However, the alpha-MHC isoform can be detected in healthy human ventricle and appears to be significantly downregulated in failing hearts. The unique biochemical properties of the alpha-MHC isoform might offer functional advantages in a failing heart that is expressing only the beta-MHC isoform. This hypothesis cannot be tested in mice or rats because both species express alpha-MHC as the predominant isoform. METHODS AND RESULTS: To test the effects of persistent alpha-MHC expression on the background of beta-MHC, we made transgenic (TG) rabbits that expressed rabbit alpha-MHC cDNA in the ventricle so that the endogenous myosin was partially replaced by the transgenically encoded species. Molecular, histological, and functional analyses showed no significant baseline effects in the TG rabbits compared with nontransgenic (NTG) littermates. To determine whether alpha-MHC expression afforded any advantages to stressed myocardium, a cohort of TG and NTG rabbits was subjected to rapid ventricular pacing. Although both the TG and NTG rabbits developed dilated cardiomyopathy, the TG rabbits had a higher shortening fraction, less septal thinning, and more normal +/-dP/dt than paced NTG rabbits. CONCLUSIONS: Transgenic expression of alpha-MHC does not have any apparent detrimental effects under basal conditions and is cardioprotective in experimental tachycardia-induced cardiomyopathy.

Charged residue alterations in the inner-core domain and carboxy-terminus of alpha-tropomyosin differentially affect mouse cardiac muscle contractility.

Two important charge differences between the alpha- and beta-tropomyosin (TM) isoforms are the exchange of a serine residue in the inner-core region at position 229, and a histidine residue at the carboxy-terminal end at position 276, with glutamic acid and asparagine, respectively. We have recently shown that altering these two residues in alpha-TM to their beta-TM counterparts in transgenic (TG) mouse hearts causes a depression in both +dP/dt and -dP/dt and a decrease in calcium sensitivity. In this study, we address whether independent charge changes at these two residues in alpha-TM modulate cardiac function differentially. To test this hypothesis we generated two TG lines: alpha-TMSer229Glu and alpha-TMHis276Asn. Molecular analyses show that 98% of native alpha-TM is replaced by mutated protein in alpha-TM229 hearts whereas alpha-TM276 hearts show 82% replacement with the mutated protein. Isolated working heart data show that alpha-TM229 TG hearts exhibit a significant decrease in both +dP/dt (7%) and -dP/dt (8%) compared with nontransgenics (NTGs) and time to peak pressure (TPP) is also reduced in alpha-TM229 hearts. alpha-TM276 hearts show a decrease only in -dP/dt (14%) and TPP is increased. pCa(2+)-tension relationships in skinned fibre preparations indicate decreased calcium sensitivity in alpha-TM229 but no change in alpha-TM276 preparations. Force-[Ca(2+)](IC) measurements from intact papillary fibres indicate that alpha-TM276 fibres produce more force per given [Ca(2+)](IC) when compared to NTG fibres, while alpha-TM229 fibres produce less force per given [Ca(2+)](IC). These data demonstrate that changing charged residues at either the inner-core domain or the carboxyl end of TM alters sarcomeric performance differently, suggesting that the function of TM is compartmentalized along its length.

Disruption of Rho signaling results in progressive atrioventricular conduction defects while ventricular function remains preserved.

Recent studies suggest that RhoA and Rac1 mediate hypertrophic signals in cardiac myocyte hypertrophy. However, effects on cardiac function caused by inhibition of their activity in the heart have yet to be evaluated. Cardiac-specific inhibition of Rho family protein activities was achieved by expressing Rho GDIalpha, an endogenous specific GDP dissociation inhibitor for Rho family proteins, using the alpha-myosin heavy-chain promoter. Increased expression of Rho GDIalpha led to atrial arrhythmias and mild ventricular hypertrophy in adult mice (4-7 months). However, left ventricular systolic and diastolic function was largely preserved before and after the development of cardiac hypertrophy, indicating that Rho GTPases are not required to maintain ventricular contractile function under basal physiological condition. Electrocardiography and intracardiac electrophysiological studies revealed first-degree atrioventricular (AV) block in the transgenic heart at 1 week of age, which further progressed into second-degree AV block at 4 weeks of age before the development of cardiac hypertrophy. Expression of connexin 40 dramatically decreased from 1 week to 4 weeks of age in the transgenic heart, which may contribute in part to the conduction defects in the transgenic mice. This study provides novel evidence for an important role of Rho GTPases in regulating AV conduction.

PKC-alpha regulates cardiac contractility and propensity toward heart failure.

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-alpha as a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-alpha on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.

Charged residue changes in the carboxy-terminus of alpha-tropomyosin alter mouse cardiac muscle contractility.

Striated muscle tropomyosin (TM) is an essential thin filament protein that is sterically and allosterically involved in calcium-mediated cardiac contraction. We have previously shown that overexpressing the beta-TM isoform in mouse hearts leads to physiological changes in myocardial relaxation and Ca(2+) handling of myofilaments. Two important charge differences in beta-TM compared to alpha-TM are the exchange of serine and histidine at positions 229 and 276 with glutamic acid and asparagine, respectively, imparting a more negative charge to beta-TM relative to alpha-TM. Our hypothesis is that the net charge at specific sites on TM might be a major determinant of its role in modulating cardiac muscle performance and in regulating Ca(2+) sensitivity of the myofilaments. To address this, we generated transgenic (TG) double mutation mouse lines (alpha-TM DM) expressing mutated alpha-TM at the two residues that differ between alpha- and beta-TM (Ser229Glu + His276Asn). Molecular analyses show 60-88% of the native TM is replaced with alpha-TM DM in the different TG lines. Work-performing heart analyses show that alpha-TM DM mouse hearts exhibit decreased rates of pressure development and relaxation (+dP/dt and -dP/dt). Skinned myofibre preparations from the TG hearts indicate a decrease in calcium sensitivity of steady state force. Protein modelling studies show that these two charge alterations in alpha-TM cause a change in the surface charges of the molecule. Our results provide the first evidence that charge changes at the carboxy-terminal of alpha-TM alter the functional characteristics of the heart at both the whole organ and myofilament levels.

Calcineurin Abeta gene targeting predisposes the myocardium to acute ischemia-induced apoptosis and dysfunction.

Cardiovascular disease is the leading cause of mortality and morbidity within the industrialized nations of the world, with coronary heart disease (CHD) accounting for as much as 66% of these deaths. Acute myocardial infarction is a typical sequelae associated with long-standing coronary heart disease resulting in large scale loss of ventricular myocardium through both apoptotic and necrotic cell death. In this study, we investigated the role that the calcium calmodulin-activated protein phosphatase calcineurin (PP2B) plays in modulating cardiac apoptosis after acute ischemia-reperfusion injury to the heart. Calcineurin Abeta gene-targeted mice showed a greater loss of viable myocardium, enhanced DNA laddering and TUNEL, and a greater loss in functional performance compared with strain-matched wild-type control mice after ischemia-reperfusion injury. RNA expression profiling was performed to uncover potential mechanisms associated with this loss of cardioprotection. Interestingly, calcineurin Abeta-/- hearts were characterized by a generalized downregulation in gene expression representing approximately 6% of all genes surveyed. Consistent with this observation, nuclear factor of activated T cells (NFAT)-luciferase reporter transgenic mice showed reduced expression in calcineurin Abeta-/- hearts at baseline and after ischemia-reperfusion injury. Finally, expression of an activated NFAT mutant protected cardiac myocytes from apoptotic stimuli, whereas directed inhibition of NFAT augmented cell death. These results represent the first genetic loss-of-function data showing a prosurvival role for calcineurin-NFAT signaling in the heart.