Lumican deficiency results in cardiomyocyte hypertrophy with altered collagen assembly

The ability of the heart to adapt to increased stress is dependent on the modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest that LUM deficient mice (lum^−/−) exhibit skin anomalies consistent with Ehlers–Danlos syndrome, lum^−/− hearts have not been evaluated. These studies show that LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum^−/− neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum^−/− hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum^−/− hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum^−/− hearts. There was also a reduction in the β and γ forms of collagenα1(I) in lum^−/− hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum^−/− hearts, indicating that LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate multiple factors of collagen assembly in the murine heart. Further investigation into the role of LUM may yield novel therapeutic targets and/or biomarkers for patients with cardiovascular disease.     

Mechanical unloading of the rat heart involves marked changes in the protein kinase–phosphatase balance

Mechanical unloading of failing hearts by left ventricular (LV) assist devices is regularly used as a bridge to transplantation and may lead to symptomatic improvement. The latter has been associated with altered phosphorylation of cardiac regulatory proteins, but the underlying mechanisms remained unknown. Here, we tested whether cardiac unloading alters protein phosphorylation by affecting the corresponding kinase–phosphatase balance. Cardiac unloading and reduction in LV mass were induced by heterotopic heart transplantation in rats for two weeks (n = 8). Native in situ hearts from the recipient animals were used as controls (n = 8). The steady-state protein kinase A (PKA) and/or Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) phosphorylation levels of phospholamban (PLB, Ser¹⁶ and Thr¹⁷) and troponin I (TnI, Ser^23/24) were decreased by 40–60% in unloaded hearts. Consistently, in these hearts PKA activity was decreased by ∼ 80% and the activity of protein phosphatase 1 and 2A was increased by 50% and 90%, respectively. In contrast, CaMKII activity was ∼ 60% higher, which may serve as a partial compensation. These data indicate that unloading shifts the kinase–phosphatase balance towards net dephosphorylation of PLB and TnI. This shift may also contribute to the reduction in phosphorylation levels of cardiac phosphoproteins observed in diseased human hearts after LVAD.     

Deletion of the β2-adrenergic receptor prevents the development of cardiomyopathy in mice

Beta adrenergic receptor (β-AR) subtypes act through diverse signaling cascades to modulate cardiac function and remodeling. Previous in vitro studies suggest that β1-AR signaling is cardiotoxic whereas β2-AR signaling is cardioprotective, and may be the case during ischemia/reperfusion in vivo. The objective of this study was to assess whether β2-ARs also play a cardioprotective role in the pathogenesis of non-ischemic forms of cardiomyopathy. To dissect the role of β1 vs β2-ARs in modulating MLP (Muscle LIM Protein) cardiomyopathy, we crossbred MLP −/− with β1 −/− or β2 −/− mice. Deletion of the β2-AR improved survival, cardiac function, exercise capacity and myocyte shortening; by contrast haploinsufficency of the β1-AR reduced survival. Pathologic changes in Ca^2 + handling were reversed in the absence of β2-ARs: peak Ca^2 + and SR Ca^2 + were decreased in MLP −/− and β1 +/−/MLP −/− but restored in β2 −/− MLP −/−. These changes were associated with reversal of alterations in troponin I and phospholamban phosphorylation. Gi inhibition increased peak and baseline Ca^2 +, recapitulating changes observed in the β2 −/−/MLP −/−. The L-type Ca^2 + blocker verapamil significantly decreased cardiac function in β2 −/− MLP −/− vs WT. We next tested if the protective effects of β2-AR ablation were unique to the MLP model using TAC-induced heart failure. Similar to MLP, β2 −/− mice demonstrated delayed progression of heart failure with restoration of myocyte shortening and peak Ca^2 + and Ca^2 + release. Deletion of β2-ARs prevents the development of MLP −/− cardiomyopathy via positive modulation of Ca^2 + due to removal of inhibitory Gi signaling and increased phosphorylation of troponin I and phospholamban. Similar effects were seen after TAC. Unlike previous models where β2-ARs were found to be cardioprotective, in these two models, β2-AR signaling appears to be deleterious, potentially through negative regulation of Ca^2 + dynamics.     

Mitochondrial DNA damage and repair during ischemia–reperfusion injury of the heart

Ischemia–reperfusion (IR) injury of the heart generates reactive oxygen species that oxidize macromolecules including mitochondrial DNA (mtDNA). The 8-oxoguanine DNA glycosylase (OGG1) works synergistically with MutY DNA glycosylase (MYH) to maintain mtDNA integrity. Our objective was to study the functional outcome of lacking the repair enzymes OGG1 and MYH after myocardial IR and we hypothesized that OGG1 and MYH are important enzymes to preserve mtDNA and heart function after IR. Ex vivo global ischemia for 30 min followed by 10 min of reperfusion induced mtDNA damage that was removed within 60 min of reperfusion in wild-type mice. After 60 min of reperfusion the ogg1^−/− mice demonstrated increased mtDNA copy number and decreased mtDNA damage removal suggesting that OGG1 is responsible for removal of IR-induced mtDNA damage and copy number regulation. mtDNA damage was not detected in the ogg1^−/−/myh^−/−, inferring that adenine opposite 8-oxoguanine is an abundant mtDNA lesion upon IR. The level and integrity of mtDNA were restored in all genotypes after 35 min of regional ischemia and six week reperfusion with no change in cardiac function. No consistent upregulation of other mitochondrial base excision repair enzymes in any of our knockout models was found. Thus repair of mtDNA oxidative base lesions may not be important for maintenance of cardiac function during IR injury in vivo. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease."     

STIM1 elevation in the heart results in aberrant Ca2 + handling and cardiomyopathy

Stromal interaction molecule 1 (STIM1) is a Ca^2 + sensor that partners with Orai1 to elicit Ca^2 + entry in response to endoplasmic reticulum (ER) Ca^2 + store depletion. While store-operated Ca^2 + entry (SOCE) is important for maintaining ER Ca^2 + homeostasis in non-excitable cells, it is unclear what role it plays in the heart, although STIM1 is expressed in the heart and upregulated during disease. Here we analyzed transgenic mice with STIM1 overexpression in the heart to model the known increase of this protein in response to disease. As expected, STIM1 transgenic myocytes showed enhanced Ca^2 + entry following store depletion and partial co-localization with the type 2 ryanodine receptor (RyR2) within the sarcoplasmic reticulum (SR), as well as enrichment around the sarcolemma. STIM1 transgenic mice exhibited sudden cardiac death as early as 6 weeks of age, while mice surviving past 12 weeks of age developed heart failure with hypertrophy, induction of the fetal gene program, histopathology and mitochondrial structural alterations, loss of ventricular functional performance and pulmonary edema. Younger, pre-symptomatic STIM1 transgenic mice exhibited enhanced pathology following pressure overload stimulation or neurohumoral agonist infusion, compared to controls. Mechanistically, cardiac myocytes isolated from STIM1 transgenic mice displayed spontaneous Ca^2 + transients that were prevented by the SOCE blocker SKF-96365, increased L-type Ca^2 + channel (LTCC) current, and enhanced Ca^2 + spark frequency. Moreover, adult cardiac myocytes from STIM1 transgenic mice showed both increased diastolic Ca^2 + and maximal transient amplitude but no increase in total SR Ca^2 + load. Associated with this enhanced Ca^2 + profile was an increase in cardiac nuclear factor of activated T-cells (NFAT) and Ca^2 +/calmodulin-dependent kinase II (CaMKII) activity. We conclude that STIM1 has an unexpected function in the heart where it alters communication between the sarcolemma and SR resulting in greater Ca^2 + flux and a leaky SR compartment.     

Prediction clinical profile to distinguish between systolic and diastolic heart failure in hospitalized patients

BackgroundIn recent decades, the growing incidence of patients with heart failure who have preserved systolic function, underlines the need to differentiate between heart failure due to diastolic dysfunction and that due to systolic dysfunction.ObjectiveTo develop a prediction profile of clinical parameters that enables clinicians to differentiate between patients with systolic and diastolic heart failure.Methods164 patients admitted for congestive heart failure to the cardiology department of an academic tertiary care hospital, whose left ventricular systolic and diastolic function had been evaluated echocardiographically and who satisfied the Framingham criteria for heart failure, were prospectively recruited. All patients answered a questionnaire which included, in addition to other clinical variables, the Framingham criteria.ResultsPatients with diastolic heart failure (61.6%) were more likely to be older, female, and to present left ventricular hypertrophy (LVH), with a lower proportion of smokers, alcohol drinkers, coronary disease, q wave and left bundle branch block (all p < 0.005). The predicting model obtained on the logistic regression analysis was very significant, with three variables and 72.3% of correct predictions (x² value = 40,457, p < 0.001). These three variables, predictors of diastolic as opposed to systolic heart failure, were female sex (OR = 3.546), left ventricle hypertrophy (OR = 4.011) and absence of coronary disease (OR = 3.547).ConclusionThree variables which can be easily evaluated, female sex, left ventricular hypertrophy and presence or absence of coronary disease, may enable clinicians to differentiate between patients with systolic or diastolic heart failure.     

Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation

Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca^2 +-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca^2 +-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca^2 +. These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β.     

Histone-deacetylase inhibition reverses atrial arrhythmia inducibility and fibrosis in cardiac hypertrophy independent of angiotensin

Atrial fibrosis influences the development of atrial fibrillation (AF), particularly in the setting of structural heart disease where angiotensin-inhibition is partially effective for reducing atrial fibrosis and AF. Histone-deacetylase inhibition reduces cardiac hypertrophy and fibrosis, so we sought to determine if the HDAC inhibitor trichostatin A (TSA) could reduce atrial fibrosis and arrhythmias. Mice over-expressing homeodomain-only protein (HopX^Tg), which recruits HDAC activity to induce cardiac hypertrophy were investigated in 4 groups (aged 14–18 weeks): wild-type (WT), HopX^Tg, HopX^Tg mice treated with TSA for 2 weeks (TSA-HopX) and wild-type mice treated with TSA for 2 weeks (TSA-WT). These groups were characterized using invasive electrophysiology, atrial fibrosis measurements, atrial connexin immunocytochemistry and myocardial angiotensin II measurements. Invasive electrophysiologic stimulation, using the same attempts in each group, induced more atrial arrhythmias in HopX^Tg mice (48 episodes in 13 of 15 HopX^Tg mice versus 5 episodes in 2 of 15 TSA-HopX mice, P < 0.001; versus 9 episodes in 2 of 15 WT mice, P < 0.001; versus no episodes in any TSA-WT mice, P < 0.001). TSA reduced atrial arrhythmia duration in HopX^Tg mice (1307 ± 289 ms versus 148 ± 110 ms, P < 0.01) and atrial fibrosis (8.1 ± 1.5% versus 3.9 ± 0.4%, P < 0.001). Atrial connexin40 was lower in HopX^Tg compared to WT mice, and TSA normalized the expression and size distribution of connexin40 gap junctions. Myocardial angiotensin II levels were similar between WT and HopX^Tg mice (76.3 ± 26.0 versus 69.7 ± 16.6 pg/mg protein, P = NS). Therefore, it appears HDAC-inhibition reverses atrial fibrosis, connexin40 remodeling and atrial arrhythmia vulnerability independent of angiotensin II in cardiac hypertrophy.     

Normal cardiac contraction in mice lacking the proline–alanine rich region and C1 domain of cardiac myosin binding protein C

Cardiac myosin binding protein C (cMyBP-C) is an essential regulator of cross bridge cycling. Through mechanisms that are incompletely understood the N-terminal domains (NTDs) of cMyBP-C can activate contraction even in the absence of calcium and can also inhibit cross bridge kinetics in the presence of calcium. In vitro studies indicated that the proline–alanine rich (p/a) region and C1 domain are involved in these processes, although effects were greater using human proteins compared to murine proteins (Shaffer et al. J Biomed Biotechnol 2010, 2010: 789798). We hypothesized that the p/a and C1 region are critical for the timing of contraction. In this study we tested this hypothesis using a mouse model lacking the p/a and C1 region (p/a-C1^−/− mice) to investigate the in vivo relevance of these regions on cardiac performance.Surprisingly, hearts of adult p/a-C1^−/− mice functioned normally both on a cellular and whole organ level. Force measurements in permeabilized cardiomyocytes from adult p/a-C1^−/− mice and wild type (Wt) littermate controls demonstrated similar rates of force redevelopment both at submaximal and maximal activation. Maximal and passive force and calcium sensitivity of force were comparable between groups as well. Echocardiograms showed normal isovolumetric contraction times, fractional shortening and ejection fraction, indicating proper systolic function in p/a-C1^−/− mouse hearts. p/a-C1^−/− mice showed a slight but significant reduction in isovolumetric relaxation time compared to Wt littermates, yet this difference disappeared in older mice (7–8 months of age). Moreover, stroke volume was preserved in p/a-C1^−/− mice, corroborating sufficient time for normal filling of the heart. Overall, the hearts of p/a-C1^−/− mice showed no signs of dysfunction even after chronic stress with an adrenergic agonist.Together, these results indicate that the p/a region and the C1 domain of cMyBP-C are not critical for normal cardiac contraction in mice and that these domains have little if any impact on cross bridge kinetics in mice. These results thus contrast with in vitro studies utilizing proteins encoding the human p/a region and C1 domain. More detailed insight in how individual domains of cMyBP-C function and interact, across species and over the wide spectrum of conditions in which the heart has to function, will be essential to a better understanding of how cMyBP-C tunes cardiac contraction.     

Carbonic anhydrase XII in valve interstitial cells promotes the regression of calcific aortic valve stenosis

AimsCalcific aortic valve stenosis (CAVS) is the most common heart valve disease. In the present work we sought to determine the reversibility of mineralization in the aortic valve.Methods and resultsBy using in vitro analyses we found that valve interstitial cells (VICs) have the ability to resorb minerals. We documented that agonist of P2Y2 receptor (P2Y2R) promoted the expression of carbonic anhydrase XII (CAXII) at the cell membrane of VICs, whereby minerals are resorbed. P2Y2R-mediated mineral resorption was corroborated by using mouse VICs isolated from wild type and P2Y2R^−/− mice. Measurements of extracellular pH (pHe) by using core–shell nanosensors revealed that P2Y2R-mediated CAXII export to the cell membrane led to an acidification of extracellular space, whereby minerals are resorbed. In vivo, we next treated LDLR^−/−/ApoB^100/100/IGF2 mice, which had developed CAVS under a high-fat/high-sucrose diet for 8 months, with 2-thioUTP (a P2Y2R agonist) or saline for the next 2 months. The administration of 2-thioUTP (2 mg/kg/day i.p.) reduced the mineral volume in the aortic valve measured with serial microCT analyses, which improved hemodynamics and reduced left ventricular hypertrophy (LVH). Examination of leaflets at necropsy confirmed a lower level of mineralization and fibrosis along with higher levels of CAXII in mice under 2-thioUTP. In another series of experiment, the administration of acetazolamide (a CA inhibitor) prevented the acidification of leaflets and the regression of CAVS induced by 2-thioUTP in LDLR^−/−/ApoB^100/100/IGF2 mice.ConclusionP2Y2R-mediated expression of CAXII by VICs acidifies the extracellular space and promotes the regression of CAVS.     

Ca2 + Sparks and Ca2 + waves are the subcellular events underlying Ca2 + overload during ischemia and reperfusion in perfused intact hearts

Abnormal intracellular Ca²⁺ cycling plays a key role in cardiac dysfunction, particularly during the setting of ischemia/reperfusion (I/R). During ischemia, there is an increase in cytosolic and sarcoplasmic reticulum (SR) Ca²⁺. At the onset of reperfusion, there is a transient and abrupt increase in cytosolic Ca^2+ +, which occurs timely associated with reperfusion arrhythmias. However, little is known about the subcellular dynamics of Ca²⁺ increase during I/R, and a possible role of the SR as a mechanism underlying this increase has been previously overlooked. The aim of the present work is to test two main hypotheses:  (1) An increase diastolic Ca²⁺ sparks frequency (cspf) constitutes a mayor substrate for the ischemia-induced diastolic Ca²⁺ increase; (2) an increase in cytosolic Ca²⁺ pro-arrhythmogenic events (Ca²⁺ waves), mediates the abrupt diastolic Ca²⁺ rise at the onset of reperfusion. We used confocal microscopy on mouse intact hearts loaded with Fluo-4. Hearts were submitted to global I/R (12/30 min) to assess epicardial Ca²⁺ sparks in the whole heart. Intact heart sparks were faster than in isolated myocytes whereas cspf was not different. During ischemia, cspf significantly increased relative to preischemia (2.07 ± 0.33 vs. 1.13 ± 0.20 sp/s/100 μm, n = 29/34, 7 hearts). Reperfusion significantly changed Ca²⁺ sparks kinetics, by prolonging Ca²⁺ sparks rise time and decreased cspf. However, it significantly increased Ca²⁺ wave frequency relative to ischemia (0.71 ± 0.14 vs. 0.38 ± 0.06 w/s/100 μm, n = 32/33, 7 hearts). The results show for the first time the assessment of intact perfused heart Ca^2 + sparks and provides direct evidence of increased Ca²⁺ sparks in ischemia that transform into Ca²⁺ waves during reperfusion. These waves may constitute a main trigger for reperfusion arrhythmias.     

CaMKIIδ mediates β-adrenergic effects on RyR2 phosphorylation and SR Ca2 + leak and the pathophysiological response to chronic β-adrenergic stimulation

Chronic activation of Ca^2 +/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the deleterious effects of β-adrenergic receptor (β-AR) signaling on the heart, in part, by enhancing RyR2-mediated sarcoplasmic reticulum (SR) Ca^2 + leak. We used CaMKIIδ knockout (CaMKIIδ-KO) mice and knock-in mice with an inactivated CaMKII site S2814 on the ryanodine receptor type 2 (S2814A) to investigate the involvement of these processes in β-AR signaling and cardiac remodeling. Langendorff-perfused hearts from CaMKIIδ-KO mice showed inotropic and chronotropic responses to isoproterenol (ISO) that were similar to those of wild type (WT) mice; however, in CaMKIIδ-KO mice, CaMKII phosphorylation of phospholamban and RyR2 was decreased and isolated myocytes from CaMKIIδ-KO mice had reduced SR Ca^2 + leak in response to isoproterenol (ISO). Chronic catecholamine stress with ISO induced comparable increases in relative heart weight and other measures of hypertrophy from day 9 through week 4 in WT and CaMKIIδ-KO mice, but the development of cardiac fibrosis was prevented in CaMKIIδ-KO animals. A 4-week challenge with ISO resulted in reduced cardiac function and pulmonary congestion in WT, but not in CaMKIIδ-KO or S2814A mice, implicating CaMKIIδ-dependent phosphorylation of RyR2-S2814 in the cardiomyopathy, independent of hypertrophy, induced by prolonged β-AR stimulation.     

CypD−/− hearts have altered levels of proteins involved in Krebs cycle,branch chain amino acid degradation and pyruvate metabolism

Cyclophilin D (CypD) is a mitochondrial chaperone that has been shown to regulate the mitochondrial permeability transition pore (MPTP). MPTP opening is a major determinant of mitochondrial dysfunction and cardiomyocyte death during ischemia/reperfusion (I/R) injury. Mice lacking CypD have been widely used to study regulation of the MPTP, and it has been shown recently that genetic depletion of CypD correlates with elevated levels of mitochondrial Ca^2 +. The present study aimed to characterize the metabolic changes in CypD^−/− hearts. Initially, we used a proteomics approach to examine protein changes in CypD^−/− mice. Using pathway analysis, we found that CypD^−/− hearts have alterations in branched chain amino acid metabolism, pyruvate metabolism and the Krebs cycle. We tested whether these metabolic changes were due to inhibition of electron transfer from these metabolic pathways into the electron transport chain. As we found decreased levels of succinate dehydrogenase and electron transfer flavoprotein in the proteomics analysis, we examined whether activities of these enzymes might be altered. However, we found no alterations in their activities. The proteomics study also showed a 23% decrease in carnitine-palmitoyltransferase 1 (CPT1), which prompted us to perform a metabolomics analysis. Consistent with the decrease in CPT1, we found a significant decrease in C4/Ci4, C5-OH/C3-DC, C12:1, C14:1, C16:1, and C20:3 acyl carnitines in hearts from CypD^−/− mice. In summary, CypD^−/− hearts exhibit changes in many metabolic pathways and caution should be used when interpreting results from these mice as due solely to inhibition of the MPTP.     

Calcium signaling regulates ventricular hypertrophy during development independent of contraction or blood flow

In utero interventions aimed at restoring left ventricular hemodynamic forces in fetuses with prenatally diagnosed hypoplastic left heart syndrome failed to stimulate ventricular myocardial growth during gestation, suggesting chamber growth during development may not rely upon fluid forces. We therefore hypothesized that ventricular hypertrophy during development may depend upon fundamental Ca^2 +-dependent growth pathways that function independent of hemodynamic forces. To test this hypothesis, zebrafish embryos were treated with inhibitors or activators of Ca^2 + signaling in the presence or absence of contraction during the period of chamber development. Abolishment of contractile function alone in the setting of preserved Ca^2 + signaling did not impair ventricular hypertrophy. In contrast, inhibition of L-type voltage-gated Ca^2 + influx abolished contraction and led to reduced ventricular hypertrophy, whereas increasing L-type voltage-gated Ca^2 + influx led to enhanced ventricular hypertrophy in either the presence or absence of contraction. Similarly, inhibition of the downstream Ca^2 +-sensitive phosphatase calcineurin, a known regulator of adult cardiac hypertrophy, led to reduced ventricular hypertrophy in the presence or absence of contraction, whereas hypertrophy was rescued in the absence of L-type voltage-gated Ca^2 + influx and contraction by expression of a constitutively active calcineurin. These data suggest that ventricular cardiomyocyte hypertrophy during chamber formation is dependent upon Ca^2 + signaling pathways that are unaffected by heart function or hemodynamic forces. Disruption of Ca^2 +-dependent hypertrophy during heart development may therefore represent one mechanism for impaired chamber formation that is not related to impaired blood flow.     

Characterization of the cardiac succinylome and its role in ischemia–reperfusion injury

Succinylation refers to modification of lysine residues with succinyl groups donated by succinyl-CoA. Sirtuin5 (Sirt5) is a mitochondrial NAD⁺-dependent deacylase that catalyzes the removal of succinyl groups from proteins. Sirt5 and protein succinylation are conserved across species, suggesting functional importance of the modification. Sirt5 loss impacts liver metabolism but the role of succinylation in the heart has not been explored. We combined affinity enrichment with proteomics and mass spectrometry to analyze total succinylated lysine content of mitochondria isolated from WT and Sirt5^−/− mouse hearts. We identified 887 succinylated lysine residues in 184 proteins. 44 peptides (5 proteins) occurred uniquely in WT samples, 289 (46 proteins) in Sirt5^−/− samples, and 554 (133 proteins) were common to both groups. The 46 unique proteins in Sirt5^−/− heart participate in metabolic processes such as fatty acid β-oxidation (Eci2) and branched chain amino acid catabolism, and include respiratory chain proteins (Ndufa7, 12, 13, Dhsa). We performed label-free analysis of the peptides common to WT and Sirt5^−/− hearts. 16 peptides from 9 proteins were significantly increased in Sirt5^−/− by at least 30%. The adenine nucleotide transporter 1 showed the highest increase in succinylation in Sirt5^−/− (108.4 fold). The data indicate that succinylation is widespread in the heart and enriched in metabolic pathways. We examined whether the loss of Sirt5 would impact ischemia–reperfusion (I/R) injury and we found an increase in infarct size in Sirt5^−/− hearts compared to WT littermates (68.5⁺/₋ 1.1% Sirt5^−/− vs 39.6⁺/⁻ 6.8% WT) following 20 min of ischemia and 90-min reperfusion. We further demonstrate that I/R injury in Sirt5^−/− heart is restored to WT levels by pretreatment with dimethyl malonate, a competitive inhibitor of succinate dehydrogenase (SDH), implicating alteration in SDH activity as causative of the injury.     

High-sensitivity troponin T is a prognostic marker for patients with aortic stenosis after valve replacement surgery

BackgroundAortic stenosis (AS) is recognized as a cause of sudden cardiac death. Recently, the measurement of high-sensitivity troponin T (hs-TnT) has become possible. Several studies have clarified that hs-TnT is a marker to indicate mortality of cardiovascular diseases.ObjectivesTo examine whether hs-TnT can be used as a prognostic marker to predict the operative outcome of AS.MethodsWe enrolled 60 patients with AS (mean age = 68.7 ± 9.6 years, male/female = 30/30). Cardiac catheterization and echocardiography were performed to evaluate the severity of AS. Aortic valve replacement surgery was performed in all patients. We defined major adverse cardiac events (MACE) as composite events of heart failure, fatal arrhythmia, and all causes of death.ResultsWe followed up the patients for 922 ± 800 days. Mean left ventricular ejection fraction was 60.0 ± 1.8%. Mean aortic valve area was 0.61 ± 0.03 cm². MACE occurred in 11 patients (18%), including 5 sudden cardiac deaths. We divided the patients into three groups based on the percentile of the plasma levels of hs-TnT. Kaplan–Meier curve revealed a statistically significant difference in MACE rate among the groups (log-rank test, χ² = 13.0, p = 0.002). We conducted a Cox proportional hazard analysis with a model including age, sex, estimated glomerular filtration rate, and hs-TnT tertile as explanatory variables to predict MACE. We found that hs-TnT tertile to be a significant factor to predict MACE (hazard ratio: 3.71, p = 0.03).Conclusionshs-TnT can be a prognostic marker for patients with AS after valve replacement surgery.