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The 'fight or flight' response to physiological stress involves sympathetic nervous system activation, catecholamine release and adrenergic receptor stimulation. In the heart, this induces positive inotropy, previously attributed to the β1-adrenergic receptor subtype. However, the role of the α1A-adrenergic receptor, which has been suggested to be protective in cardiac pathology, has not been investigated in the setting of physiological stress. To explore this, we developed a tamoxifen-inducible, cardiomyocyte-specific α1A-adrenergic receptor knock-down mouse model, challenged mice to four weeks of endurance swim training and assessed cardiac outcomes. With 4-OH tamoxifen treatment, expression of the α1A-adrenergic receptor was knocked down by 80-89%, without any compensatory changes in the expression of other adrenergic receptors, or changes to baseline cardiac structure and function. Swim training caused eccentric hypertrophy, regardless of genotype, demonstrated by an increase in heart weight/tibia length ratio (30% and 22% in vehicle- and tamoxifen-treated animals, respectively) and an increase in left ventricular end diastolic volume (30% and 24% in vehicle- and tamoxifen-treated animals, respectively) without any change in the wall thickness/chamber radius ratio. Consistent with physiological hypertrophy, there was no increase in fetal gene program (Myh7, Nppa, Nppb or Acta1) expression. In response to exercise-induced volume overload, stroke volume (39% and 30% in vehicle- and tamoxifen-treated animals, respectively), cardiac output/tibia length ratio (41% in vehicle-treated animals) and stroke work (61% and 33% in vehicle- and tamoxifen-treated animals, respectively) increased, regardless of genotype. These findings demonstrate that cardiomyocyte α1A-adrenergic receptors are not necessary for cardiac adaptation to endurance exercise stress and their acute ablation is not deleterious.

Pressure overload-induced cardiac hypertrophy is a maladaptive response with poor outcomes and limited treatment options. The transient receptor potential melastatin 4 (TRPM4) ion channel is key to activation of a Ca2+-calmodulin kinase II (CaMKII)-dependent hypertrophic signalling pathway after pressure overload, but TRPM4 is neither stretch-activated nor Ca2+-permeable. Here we show that Piezo1, which is both stretch-activated and Ca2+-permeable, is the mechanosensor that transduces increased myocardial forces into the chemical signal that initiates hypertrophic signalling via TRPM4. Cardiomyocyte-specific deletion of Piezo1 in adult mice prevented activation of CaMKII and inhibited the hypertrophic response: residual hypertrophy was associated with calcineurin activation in the absence of its usual inhibition by activated CaMKII. Piezo1 deletion prevented upregulation of the sodium-calcium exchanger and downregulation of the T-type calcium channel after pressure overload. These findings establish Piezo1 as the cardiomyocyte mechanosensor that instigates the maladaptive hypertrophic response to pressure overload, opening an avenue to novel therapies.

Pathological left ventricular hypertrophy (LVH) occurs in response to pressure overload and remains the single most important clinical predictor of cardiac mortality. The molecular pathways in the induction of pressure overload LVH are potential targets for therapeutic intervention. Current treatments aim to remove the pressure overload stimulus for LVH, but do not completely reverse adverse cardiac remodelling. Although numerous molecular signalling steps in the induction of LVH have been identified, the initial step by which mechanical stretch associated with cardiac pressure overload is converted into a chemical signal that initiates hypertrophic signalling remains unresolved. In this study, we show that selective deletion of transient receptor potential melastatin 4 (TRPM4) channels in mouse cardiomyocytes results in an approximately 50% reduction in the LVH induced by transverse aortic constriction. Our results suggest that TRPM4 channel is an important component of the mechanosensory signalling pathway that induces LVH in response to pressure overload and represents a potential novel therapeutic target for the prevention of pathological LVH. ispartof: ELIFE vol:10 ispartof: location:England status: published

Pathological left ventricular hypertrophy (LVH) is a consequence of pressure overload caused by systemic hypertension or aortic stenosis and is a strong predictor of cardiac failure and mortality. Understanding the molecular pathways in the development of pathological LVH may lead to more effective treatment. Here, we show that the transient receptor potential cation channel subfamily melastatin 4 (TRPM4) ion channel is an important contributor to the mechanosensory transduction of pressure overload that induces LVH. In mice with pressure overload induced by transverse aortic constriction (TAC) for two weeks, cardiomyocyte TRPM4 expression was reduced, as compared to control mice. Cardiomyocyte-specific TRPM4 inactivation reduced by ~50% the degree of TAC-induced LVH, as compared with wild type (WT). In WT mice, TAC activated the CaMKIIδ-HDAC4-MEF2A but not the calcineurin-NFAT-GATA4 pathway. In TRPM4 knock-out mice, activation of the CaMKIIδ-HDAC4-MEF2A pathway by TAC was significantly reduced. However, consistent with a reduction in the known inhibitory effect of CaMKIIδ on calcineurin activity, reduction in the CaMKIIδ-HDAC4-MEF2A pathway was associated with partial activation of the calcineurin-NFAT-GATA4 pathway. These findings indicate that the TRPM4 channel and its cognate signalling pathway are potential novel therapeutic targets for the prevention of pathological pressure overload-induced LVH.Significance statementPathological left ventricular hypertrophy (LVH) occurs in response to pressure overload and remains the single most important clinical predictor of cardiac mortality. Preventing pressure overload LVH is a major goal of therapeutic intervention. Current treatments aim to remove the stimulus for LVH by lowering elevated blood pressure or replacing a stenotic aortic valve. However, neither of these interventions completely reverses adverse cardiac remodelling. Although numerous molecular signalling steps in the induction of LVH have been identified, the initial step by which mechanical stretch associated with cardiac pressure overload is converted into a chemical signal that initiates hypertrophic signalling, remains unresolved. Here, we demonstrate that the TRPM4 channel is a component of the mechanosensory transduction pathway that ultimately leads to LVH.

RationaleGq-coupled receptors are thought to play a critical role in the induction of left ventricular hypertrophy (LVH) secondary to pressure overload, although mechano-sensitive channel activation by a variety of mechanisms has also been proposed, and the relative importance of calcineurin- and calmodulin kinase II (CaMKII)-dependent hypertrophic pathways remains controversial.ObjectiveTo determine the mechanisms regulating the induction of LVH in response to mechanical pressure overload.Methods and ResultsTransgenic mice with cardiac-targeted inhibition of Gq-coupled receptors (GqI mice) and their non-transgenic littermates (NTL) were subjected to neurohumoral stimulation (continuous, subcutaneous angiotensin II (AngII) infusion for 14 days) or mechanical pressure overload (transverse aortic arch constriction (TAC) for 21 days) to induce LVH. Candidate signalling pathway activation was examined. As expected, LVH observed in NTL mice with AngII infusion was attenuated in heterozygous (GqI+/-) mice and absent in homozygous (GqI-/-) mice. In contrast, LVH due to TAC was unaltered by either heterozygous or homozygous Gq inhibition. Gene expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and α-skeletal actin (α-SA) was increased 48 hours after AngII infusion or TAC in NTL mice; in GqI mice, the increases in ANP, BNP and α-SA in response to AngII were completely absent, as expected, but all three increased after TAC. Increased nuclear translocation of nuclear factor of activated T-cells c4 (NFATc4), indicating calcineurin pathway activation, occurred in NTL mice with AngII infusion but not TAC, and was prevented in GqI mice infused with AngII. Nuclear and cytoplasmic CaMKIIδ levels increased in both NTL and GqI mice after TAC but not AngII infusion, with increased cytoplasmic phospho- and total histone deacetylase 4 (HDAC4) and increased nuclear myocyte enhancer factor 2 (MEF2) levels.ConclusionCardiac Gq receptors and calcineurin activation are required for neurohumorally mediated LVH but are not required for LVH induced by mechanical pressure overload (TAC); the latter is mediated by activation of the CaMKII-HDAC4-MEF2 pathway.

Animal models of pressure overload are valuable for understanding hypertensive heart disease. We characterised a surgical model of pressure overload-induced hypertrophy in C57BL/6J mice produced by suprarenal aortic constriction (SAC). Compared to sham controls, at one week post-SAC systolic blood pressure was significantly elevated and left ventricular (LV) hypertrophy was evident by a 50% increase in the LV weight-to-tibia length ratio due to cardiomyocyte hypertrophy. As a result, LV end-diastolic wall thickness-to-chamber radius (h/R) ratio increased, consistent with the development of concentric hypertrophy. LV wall thickening was not sufficient to normalise LV wall stress, which also increased, resulting in LV systolic dysfunction with reductions in ejection fraction and fractional shortening, but no evidence of heart failure. Pathological LV remodelling was evident by the re-expression of fetal genes and coronary artery perivascular fibrosis, with ischaemia indicated by enhanced cardiomyocyte Hif1a expression. The expression of stem cell factor receptor, c-Kit, was low basally in cardiomyocytes and did not change following the development of robust hypertrophy, suggesting there is no role for cardiomyocyte c-Kit signalling in pathological LV remodelling following pressure overload.

The ‘fight or flight’ response, involving sympathetic nervous system activation and catecholamine release, is central to survival. Resulting activation of adrenergic receptors (ARs) drives physiological responses, such as positive inotropy, which to date has been attributed solely to β 1 -AR/Gs/adenylyl cyclase/protein kinase A pathway-activation. We previously demonstrated that overexpression of the α 1A -AR is cardioprotective in rodents in the setting of pathological cardiac stress; a finding in agreement with studies of α 1A -AR inactivation. However, it remains unknown if α 1A -ARs also protect the heart from physiological stress. Using a tamoxifen-inducible, cardiomyocyte-specific α 1A -AR knockdown mouse model, we evaluated cardiac responses to swim exercise. Treatment of adult (8-12 week) mice with 4-OH tamoxifen (Tam) resulted in a 7.4 fold decrease (p1A -AR mRNA levels, with no compensatory changes in expression of β 1 -, β 2 - or α 2 -AR subtypes. A 90 minute, twice daily, four-week swim challenge induced eccentric cardiac hypertrophy, with Tam- and vehicle-treated mice displaying similar increases in heart weight/tibia length (mean increase: 1.4 and 1.6mg/mm, respectively; pIgf-1 mRNA increased 1.7 and 0.7 fold (p=0.004 vs sedentary mice), respectively, with no increase in Nppb , Myh7 or Acta1 mRNA. Cardiac function, assessed by micromanometry and echocardiography, showed increases in dP/dt min/EDV (mean: -117 vs -150 mmHg/s/μL; p=0.011), end-diastolic volume (mean: 76.9 vs 68.0μL; p=0.038) and end-systolic volume (mean: 29.4 vs 21.9μL; p=0.018), as well as a decrease in ejection fraction (mean: 63 vs 68%; p=0.046) in Tam- compared with vehicle-treated mice, respectively. Thus, despite a similar hypertrophic response, mice with cardiomyocyte-specific α 1A -AR inactivation showed an impaired contractile response to a sustained swim challenge. These findings are consistent with cardiomyocyte α 1A -ARs playing an important role in maintaining cardiac contractility in response to physiological stress.

The loss of regenerative properties in adult cardiomyocytes (CMs) is directly linked to their inability to proliferate. Following an extensive ischaemic event in an aged heart, fibrotic scar formation is the only repair process and eventually heart failure develops. However, molecular and cellular cues in the neonatal heart support that cardiac regeneration is possible in presence of proliferating CMs. Based on previous studies demonstrating that endothelial nitric oxide synthase (eNOS) regulates proliferation in both endothelial cells (ECs) and CMs, we hypothesized that eNOS signaling could play a cardioprotective role. To test our hypothesis, we injected different combinations of co-cultured ECs and CMs in the LV muscle wall of MI mice (permanent LAD ligation). First, injected cells were isolated from either WT or KO eNOS neonatal mice and then co-cultured to form 3D vascularized cardiac spheroids (VCSs), which were eventually transplanted in adult MI mice on the day of the procedure. Control infarcted animals received media-only (vehicle). Other mice received a suspension of co-cultured VCSs in media as follows: i ) WT CMs and ECs; ii ) WT CMs and KO ECs; iii ) KO CMs and WT ECs. Following 28 days, injection of WT cells increased the ejection fraction (EF%) by 20% compared with control animals (61%±4% and 41%±11%, respectively). When eNOS was absent in either CMs or ECs, the EF% was 40%±5% and 46%±2%, respectively, suggesting that the eNOS-mediated protection is dependent on its presence in both cells. Histological analyses confirmed the presence of WT VCSs in MI mice, contributing to a thicker wall thickness compared to vehicle MI mice. No VCSs were observed in the LV wall when KO cells were injected. Therefore, our results strongly suggest that eNOS may play a major role via both an autocrine (CMs) and paracrine (ECs) mechanism. Current studies are focusing on further evaluating the mechanism(s) for this eNOS-mediated protective role. To our knowledge, this is the first study combining cellular and genetic approaches to evaluate the cardioprotective role of eNOS in the heart. A better understanding of this mechanisms may have significant impact for the development of improved molecular and cell therapeutics (including stem cells) for heart failure patients.

Congenital heart disease (CHD) is the most common type of birth defect. In recent years, research has focussed on identifying the genetic causes of CHD. However, only a minority of CHD cases can be attributed to single gene mutations. In addition, studies have identified different environmental stressors that promote CHD, but the additive effect of genetic susceptibility and environmental factors is poorly understood. In this context, we have investigated the effects of short-term gestational hypoxia on mouse embryos genetically predisposed to heart defects. Exposure of mouse embryos heterozygous for Tbx1 or Fgfr1/Fgfr2 to hypoxia in utero increased the incidence and severity of heart defects while Nkx2-5+/− embryos died within 2 days of hypoxic exposure. We identified the molecular consequences of the interaction between Nkx2-5 and short-term gestational hypoxia, which suggest that reduced Nkx2-5 expression and a prolonged hypoxia-inducible factor 1α response together precipitate embryo death. Our study provides insight into the causes of embryo loss and variable penetrance of monogenic CHD, and raises the possibility that cases of foetal death and CHD in humans could be caused by similar gene-environment interactions.

Echocardiography is an invaluable tool for characterizing cardiac structure and function in vivo. Technological advances in high-frequency ultrasound over the past 3 decades have increased spatial and temporal resolution, and facilitated many important clinical and basic science discoveries. Successful reverse translation of established echocardiographic techniques, including M-mode, B-mode, color Doppler, pulsed-wave Doppler, tissue Doppler and, most recently, myocardial deformation imaging, from clinical cardiology into the basic science laboratory has enabled researchers to achieve a deeper understanding of myocardial phenotypes in health and disease. With high-frequency echocardiography, detailed evaluation of ventricular systolic function in a range of small animal models is now possible. Furthermore, improvements in frame rate and the advent of diastolic strain rate imaging, when coupled with the use of select pulsed-wave Doppler parameters, such as isovolumic relaxation time and E wave deceleration, have enabled nuanced interpretation of ventricular diastolic function. Comparing pulsed-wave Doppler indices of atrioventricular inflow during early and late diastole with parameters that describe the simultaneous myocardial deformation (e.g., tissue Doppler e and a, global longitudinal strain rate and global longitudinal velocity) may yield additional insights related to myocardial compliance. This review will provide a historical perspective of the development of high-frequency echocardiography and consider how ongoing innovation will help future-proof this important imaging modality for 21st century translational research.

We have previously demonstrated that adult transgenic C57BL/6J mice with CM-restricted overexpression of the dominant negative W v mutant protein (dn-c-kit-Tg) respond to pressure overload with robust cardiomyocyte (CM) cell cycle entry. Here, we tested if outcomes after myocardial infarction (MI) due to coronary artery ligation are improved in this transgenic model. Compared to non-transgenic littermates (NTLs), adult male dn-c-kit-Tg mice displayed CM hypertrophy and concentric left ventricular (LV) hypertrophy in the absence of an increase in workload. Stroke volume and cardiac output were preserved and LV wall stress was markedly lower than that in NTLs, leading to a more energy-efficient heart. In response to MI, infarct size in adult (16-week old) dn-c-kit-Tg hearts was similar to that of NTL after 24 h but was half that in NTL hearts 12 weeks post-MI. Cumulative CM cell cycle entry was only modestly increased in dn-c-kit-Tg hearts. However, dn-c-kit-Tg mice were more resistant to infarct expansion, adverse LV remodelling and contractile dysfunction, and suffered no early death from LV rupture, relative to NTL mice. Thus, pre-existing cardiac hypertrophy lowers wall stress in dn-c-kit-Tg hearts, limits infarct expansion and prevents death from myocardial rupture.

SUMMARYThe interstitial and perivascular spaces of the mammalian heart contain a highly interactive tissue community essential for cardiac homeostasis, repair and regeneration. Mesenchymal cells (fibroblasts) are one of the most abundant cell types, playing key roles as sentinels, tissue architects, paracrine signaling hubs and lineage precursors, and are linked to heart disease through their roles in inflammation and fibrosis. Platelet-derived growth factors (PDGFs) are secreted by several cell types involved in cardiac injury and repair, and are recognized mitogens for cardiac fibroblasts and mesenchymal stem cells. However, their roles are complex and investigations of their impact on heart repair have produced contrasting outcomes, leaving therapeutic potential uncertain. Here, we use new approaches and tools, including single cell RNA sequencing, to explore cardiac fibroblast heterogeneity and how PDGF receptor α (PDGFRα) signaling impacts fibroblasts during heart repair. Short-term systemic delivery of PDGF-AB to mice from the time of myocardial infarction (MI) led to enhanced anatomical and functional recovery. Underpinning these benefits was a priming effect, in which PDGF-AB accelerated exit of fibroblasts from quiescence and induced a higher translational biosynthetic capacity in both fibroblasts and macrophages without triggering fibrosis. Our study highlights the significant biosynthetic heterogeneity and plasticity in cardiac fibroblast populations, and suggests a rationale for a novel therapeutic approach to cardiac injury involving controlled stimulation of fibroblast activation.

Lamin A/C mutations are the most common cause of familial dilated cardiomyopathy (DCM) but the pathogenetic mechanisms are incompletely understood. Nesprins are spectrin repeat-containing proteins that interact with lamin A/C and are components of the linker-of-nucleoskeleton-and-cytoskeleton (LINC) complex that connects the nuclear envelope to the actin cytoskeleton. Our aim was to determine whether changes in nesprin-1 and actin might contribute to DCM in homozygous Lmna knockout ( Lmna −/− ) mice. Here we find that Lmna −/ − cardiomyocytes have altered nuclear envelope morphology, disorganization of nesprin-1 and heterogeneity in the distribution of nuclear and cytoskeletal actin. Functional interactions of nesprin-1 with nuclear G-actin and with the cytoskeletal γ-actin, α-cardiac actin and α-smooth muscle actin (α-SMA) isoforms were shown by immunoprecipitation and Western blotting. At 4–6 weeks of age, Lmna −/ − mice had normal levels of γ-actin and α-cardiac actin, but α-SMA expression was increased by 50%. In contrast to the predominant vascular distribution of α-SMA in WT ventricular sections, α-SMA had a diffuse staining pattern in Lmna −/ − sections. Osmotic swelling studies showed enhanced radial swelling in Lmna −/ − cardiomyocytes indicative of cytoskeletal instability. The distensibility of Lmna −/ − cardiomyocytes with osmotic stress was reduced by addition of α-SMA-specific fusion peptide. Our findings support a model in which uncoupling of the nucleus and cytoskeleton associated with disruption of the LINC complex promotes mechanical instability and defective force transmission in cardiomyocytes. Changes in the distribution and expression patterns of nuclear and cytoskeletal actin suggest that diverse transcriptional and structural defects may also contribute to DCM in Lmna −/ − mice.

Redox signaling has been implicated in the loss of proliferative/regenerative ability of cardiomyocytes (CMs) after birth. This is one of the main reasons why a mature, aged heart cannot regenerate following an extensive inschemic event, such as myocardial infarction (MI). Based on our previous studies on the roles played by endothelial nitric oxide synthase (eNOS) on endothelial cell (EC) and CM proliferation, we hypothesized that eNOS signaling could play a protective role against MI. For this, we used an MI mouse model (permanent LAD ligation). These mice were injected in the muscle wall with different combinations of ECs and CMs isolated from either WT or KO eNOS neonatal mice. Cells were first co-cultured in hanging drops for ~5 days until formation of 3D vascularized cardiac spheroids (VCSs) and then transplanted in adult MI mice. Infarcted animals received either vehicle (media) or a suspension of VCSs in media. Adult mice received VCSs generated by co-culturing either: i) WT CMs and WT ECs; or ii) WT CMs and KO ECs; or iii) KO CMs and WT ECs. Our analysis 28 days following the procedure showed that the higher ejection fraction measured in VCS-injected mice compared with animals receiving media-only (61% ± 4% and 41% ± 11%, respectively) was dependent on eNOS presence in both ECs and CMs. Specifically, genetic deletion of eNOS in either KO ECs or KO CMs in group (ii) and (iii) reduced the ejection fraction to 40% ± 5% and 46% ± 2%, respectively. Therefore, our results strongly suggest that eNOS may play a major role via both an autocrine and paracrine mechanism. Current studies are focusing on evaluating the mechanism(s) for this eNOS-mediated protective role, possibly by promoting angiogenesis and/or proliferation, together with inhibition of inflammatory pathways. A better understanding of the eNOS-mediated protective role may have significant therapeutical impact in heart failure patients.

Rationale : Mutations in the LMNA gene, which encodes the nuclear lamina proteins lamin A and lamin C, are the most common cause of familial dilated cardiomyopathy (DCM). Mechanical stress-induced apoptosis has been proposed as the mechanism underpinning DCM in lamin A/C–deficient hearts, but supporting in vivo evidence has been lacking. Objective : Our aim was to study interventions to modify mechanical stress in heterozygous Lmna knockout ( Lmna +/− ) mice. Methods and Results : Cardiac structure and function were evaluated before and after exercise training, thoracic aortic constriction, and carvedilol treatment. Lmna +/− mice develop adult-onset DCM with relatively more severe disease in males. Lmna +/− cardiomyocytes show altered nuclear morphology and perinuclear desmin organization, with enhanced responses to hypo-osmotic stress indicative of cytoskeletal instability. Despite these structural defects that provide a template for mechanical stress-induced damage, young Lmna +/− mice subjected to 6 weeks of moderate or strenuous exercise training did not show induction of apoptosis or accelerated DCM. In contrast, regular moderate exercise attenuated DCM development in male Lmna +/− mice. Sustained pressure overload generated by thoracic aortic constriction depressed ventricular contraction in young wild-type and Lmna +/− mice with no sex or genotype differences in the time-course or severity of response. Treatment of male Lmna +/− mice from 12 to 40 weeks with the β-blocker, carvedilol, prevented the dilatation and contractile dysfunction that was observed in placebo-treated mice. Conclusions : These data suggest that factors other than mechanical stress-induced apoptosis contribute to DCM and provide the first demonstration that regular moderate exercise and carvedilol can modify disease progression in lamin A/C–deficient hearts.

Mutations in the giant sarcomeric protein titin (TTN) are a major cause for inherited forms of dilated cardiomyopathy (DCM). We have previously developed a mouse model that imitates a TTN truncation mutation we found in a large pedigree with DCM. While heterozygousTtnknock-in mice do not display signs of heart failure under sedentary conditions, they recapitulate the human phenotype when exposed to the pharmacological stressor angiotensin II or isoproterenol. In this study we investigated the effects of pressure overload by transverse aortic constriction (TAC) in heterozygous (Het)Ttnknock-in mice. Two weeks after TAC, Het mice developed marked impairment of left ventricular ejection fraction(p<0.05), while wild-type (WT) TAC mice did not. Het mice also trended toward increased ventricular end diastolic pressure and volume compared to WT littermates. We found an increase in histologically diffuse cardiac fibrosis in Het compared to WT in TAC mice. This study shows that a pattern of DCM can be induced by TAC-mediated pressure overload in a TTN-truncated mouse model. This model enlarges our arsenal of cardiac disease models, adding a valuable tool to understand cardiac pathophysiological remodeling processes and to develop therapeutic approaches to combat heart failure.

Background. The aim of this study was to determine the efficacy of cariporide (a sodium-hydrogen exchanger inhibitor), BMS180448 (a pharmacologic ischemic preconditioning agent), and the combination thereof, as adjuvant therapies for extended cardiac allograft preservation. Methods. A porcine model of donor brain death and orthotopic heart transplantation was used. All hearts were arrested and stored for 14 hr in an extracellular preservation solution. Control hearts (CON; n=3) did not receive any additional treatment. Treated hearts received BMS180448 alone (BMS; n=3), cariporide alone (CAR; n=6), or both BMS180448 and cariporide (B+C; n=6). Donors of BMS180448-treated hearts received 2 mg/kg, 15 min before explantation. Donors and recipients of cariporide-treated hearts received 2 mg/kg, 15 min before explantation and reperfusion, respectively. Results. The CON and BMS arms of the study were terminated after three transplantations because initial results in these groups were poor. Significantly, none of the control hearts could be weaned successfully from bypass, whereas all of the treated hearts were weaned successfully (CAR vs. CON and B+C vs. CON:P =0.012). The rate of troponin I release during the first 3 hr after reperfusion was significantly lower in CAR (P =0.0180) and B+C (P =0.0154) recipients than in CON recipients. Mean plasma troponin I levels (μg/mL) 3 hr after reperfusion were as follows: CON 633±177, BMS 576±110, CAR 346±93, and B+C 296±97. Conclusion. In this porcine model of extended cardiac allograft preservation, cariporide was more effective than BMS180448 as an adjuvant to our usual preservation solution. There was no additional benefit from the combination of the two therapies.

Objective: To determine if the initial rate of troponin I release post-reperfusion reflects the effectiveness of myocardial protection during cardiac allograft preservation. Methods: A porcine model of orthotopic heart transplantation was used. Data from two control groups (CON4 and CON14) and two treatment groups (CAR4 and CAR14) were analysed. Hearts in CON4 (n ¼ 6) and CAR4 (n ¼ 6) were subjected to 4 h of ischaemia while hearts in CON14 (n ¼ 3) and CAR14 (n ¼ 6) were subjected to 14 h of ischaemia. All hearts were arrested and stored in the same extracellular preservation solution. Both donor and recipient animals in the CAR4 and CAR14 groups received a single intravenous dose of cariporide (2 mg/kg), prior to explantation and reperfusion, respectively. Results: Mean (SEM) plasma troponin I levels (mg/ml) 3 h postreperfusion were: CON4 210 ^ 52, CAR4 68 ^ 21, CON14 633 ^ 177, CAR14 346 ^ 93. On multiple linear regression analysis, the rate of troponin I release over the first 3 h post-reperfusion was significantly lower in hearts stored for 4 h compared to hearts stored for 14 h (P , 0:0001) and in hearts treated with cariporide compared to control hearts (P ¼ 0:0017). Early graft function was superior in hearts treated with cariporide, when compared to control hearts stored for the same period of time. All of the CAR14 hearts could be weaned from cardiopulmonary bypass whereas none of the CON14 could be weaned (6/6 vs. 0/3; P ¼ 0:012). While all hearts stored for 4 h could be weaned, contractility, as measured by the preload recruitable stroke work (PRSW) relationship, was significantly better preserved in CAR4 hearts than in CON4 hearts (P , 0:0001). Conclusions: The initial rate of troponin I release post-reperfusion is determined by the duration of cardiac allograft ischaemia. Altering the myocardial preservation strategy can reduce the rate of release. Such reductions are associated with improvements in early graft function. These findings validate the initial rate of troponin I release post-reperfusion as an end-point when comparing cardiac allograft preservation strategies. In addition, the present study provides indirect evidence that troponin I degradation during ischaemia-reperfusion is related to the accumulation of intracellular calcium. q 2003 Elsevier Science B.V. All rights reserved.

Abstract —Heterozygous mutations in the cardiac homeobox gene, NKX2-5 , underlie familial cases of atrial septal defect (ASD) with severe atrioventricular conduction block. In this study, mice heterozygous for Nkx2-5 –null alleles were assessed for analogous defects. Although ASD occurred only rarely, atrial septal dysmorphogenesis was evident as increased frequencies of patent foramen ovale and septal aneurysm, and decreased length of the septum primum flap valve. These parameters were compounded by genetic background effects, and in the 129/Sv strain, septal dysmorphogenesis bordered on ASD in 17% of Nkx2-5 heterozygotes. In a proportion of neonatal heterozygotes, as well as in adults with ASD, we found that the size of the foramen ovale was significantly enlarged and altered in shape, potentially exposing the normally thin septum primum to excessive hemodynamic forces. Therefore, defective morphogenesis of the septum secundum may be one contributing factor in the generation of patent foramen ovale, septal aneurysm, and certain ASDs. Mild prolongation of P-R interval in females and an increased frequency of stenotic bicuspid aortic valves were also features of the Nkx2-5 heterozygous phenotype. Our data demonstrate that the complex effects of Nkx2-5 haploinsufficiency in mice are weaker but convergent with those in humans. As in the mouse, the phenotype of human NKX2-5 mutations may be modulated by interacting alleles.

Background —Increased left ventricular (LV) contractile force or oxygen consumption has been documented with increased coronary arterial pressure (CAP) and flow (Gregg phenomenon). We investigated whether the increase in contractile force with increased LV afterload might be mediated by the concomitant increase in CAP when coronary autoregulation is intact. Methods and Results —The LV of 6 autonomically blocked open-chest dogs was perfused through the left main coronary artery by a cannula with a side gate to the aortic root. With the gate open, CAP increased from 77±20 to 93±20 mm Hg ( P P Conclusions —The concomitant increase in CAP with increased afterload in the intact canine LV does not contribute to the afterload-induced increase in contractile force. Coronary perfusion pressure per se does not influence LV contractile function. Coronary perfusion pressure influences contractility only when coronary flow changes.

Aims To determine the mechanisms by which the α1A-adrenergic receptor (AR) regulates cardiac contractility. Background We reported previously that transgenic mice with cardiac-restricted α1A-AR overexpression (α1A-TG) exhibit enhanced contractility but not hypertrophy, despite evidence implicating this Gαq/11-coupled receptor in hypertrophy. Methods Contractility, calcium (Ca2+) kinetics and sensitivity, and contractile proteins were examined in cardiomyocytes, isolated hearts and skinned fibers from α1A-TG mice (170-fold overexpression) and their non-TG littermates (NTL) before and after α1A-AR agonist stimulation and blockade, angiotensin II (AngII), and Rho kinase (ROCK) inhibition. Results Hypercontractility without hypertrophy with α1A-AR overexpression is shown to result from increased intracellular Ca2+ release in response to agonist, augmenting the systolic amplitude of the intracellular Ca2+ concentration [Ca2+]i transient without changing resting [Ca2+]i. In the absence of agonist, however, α1A-AR overexpression reduced contractility despite unchanged [Ca2+]i. This hypocontractility is not due to heterologous desensitization: the contractile response to AngII, acting via its Gαq/11-coupled receptor, was unaltered. Rather, the hypocontractility is a pleiotropic signaling effect of the α1A-AR in the absence of agonist, inhibiting RhoA/ROCK activity, resulting in hypophosphorylation of both myosin phosphatase targeting subunit 1 (MYPT1) and cardiac myosin light chain 2 (cMLC2), reducing the Ca2+ sensitivity of the contractile machinery: all these effects were rapidly reversed by selective α1A-AR blockade. Critically, ROCK inhibition in normal hearts of NTLs without α1A-AR overexpression caused hypophosphorylation of both MYPT1 and cMLC2, and rapidly reduced basal contractility. Conclusions We report for the first time pleiotropic α1A-AR signaling and the physiological role of RhoA/ROCK signaling in maintaining contractility in the normal heart.

We compared the effects of hormone resuscitation (HR) with a norepinephrine-based protocol on cardiac function, hemodynamics and need for vasopressor support after brain death in a porcine model. Following brain death induction, animals were treated with norepinephrine and fluids for 3 h. In the following 3 h, they continued on norepinephrine and fluids (control) or received additional HR (triiodothyronine, methylprednisolone, vasopressin, insulin). Data were collected pre-brain death, 3 and 6 h post-brain death. At 6 h, median norepinephrine use was higher in controls (0.563 vs. 0 microg/kg/min; p < 0.005), with 6/8 HR animals weaned off norepinephrine compared with 0/9 controls. Mean arterial pressure was higher in HR animals at 6 h (74 +/- 17 vs. 54 +/- 14 mmHg; p < 0.05). Cardiac contractility was also significantly higher in HR animals at 6 h (stroke work index 1.777 vs. 1.494). After collection of 6 h data, all animals were placed on the same low dose of norepinephrine. At 6.25 h, HR animals had higher stroke work (3540 +/- 1083 vs. 1536 +/- 702 mL.mmHg; p < 0.005), stroke volume (37.2 +/- 8.2 vs. 21.5 +/- 9.8 mL; p < 0.01) and cardiac output (5.8 +/- 1.4 vs. 3.2 +/- 1.2 L/min; p < 0.005). HR in a porcine model of brain death reduces norepinephrine requirements, and improves hemodynamics and cardiac function. These results support the use of HR in the management of the brain-dead donor.

Background: We aimed to develop a large animal model of orthotopic cardiac transplantation, incorporating donor brain death, to assess new methods of preservation of the donor heart. Methods: Brain death was achieved in the donor pig by inflation of a 20 cc subdural balloon 1 h prior to harvest. The donor heart was stored for 6 h with conventional hypothermic ischaemic preservation. It was then transplanted orthotopically into the recipient pig using the Lower and Shumway technique. One hour after reperfusion, the transplanted heart was weaned from cardiopulmonary bypass with dobutamine support. Dobutamine support was continued for up to 4 h, if required. After 6 h of physiological and biochemical evaluation, the recipient was euthanased and the heart excised for histological assessment. Results: All pigs experienced the classical haemodynamic changes associated with brain death. This resulted in the release of Troponin I, consistent with myocardial injury. The donor operation was successfully completed in 11 out of 13 pigs. Six out of 11 transplanted hearts were successfully weaned from cardiopulmonary bypass, but required ongoing dobutamine support. Conclusions: This porcine model of orthotopic cardiac transplantation is a relevant and practical large animal model for the assessment of new methods of preservation of the donor heart.

We previously reported that transgenic (TG) mice with cardiac-restricted alpha(1A)-adrenergic receptor (alpha(1A)-AR)-overexpression showed enhanced contractility, but no hypertrophy. Since chronic inotropic enhancement may be deleterious, we investigated if long-term, cardiac function and longevity are compromised. alpha(1A)-TG mice, but not their non-TG littermates (NTLs), showed progressive loss of left ventricular (LV) hypercontractility (dP/dt(max): 14,567+/-603 to 11,610+/-915 mmHg/s, P0.05, A1A1 line: 170-fold overexpression; and 13,625+/-826 to 8322+/-682 mmHg/s, respectively, P0.05, A1A4 line: 112-fold overexpression, at 2 and 6 months, respectively). Both TG lines developed LV fibrosis, but not LV dilatation or hypertrophy, despite activation of hypertrophic signaling pathways. Microarray and real time RT-PCR analyses revealed activation of matricellular protein genes, including those for thrombospondin 1, connective tissue growth factor and tenascin C, but not transforming growth factor beta1. Life-span was markedly shortened (mean age at death: 155 days, A1A1 line; 224 days, A1A4 line compared with NTLs:300 days). Telemetric electrocardiography revealed that death in the alpha(1A)-AR TG mice was due to cardiac standstill preceded by a progressive diminution in QRS amplitude, but not by arrhythmias. The QRS changes and sudden death could be mimicked by alpha(1)-AR activation, and reversed preterminally by alpha(1)-AR blockade, suggesting a relationship to stress- or activity-associated catecholamine release. Thus, long-term augmentation of cardiac alpha(1A)-adrenergic drive leads to premature death and progressive LV fibrosis with reactivation of matricellular protein genes. To our knowledge this is the first evidence in vivo for a role of the alpha(1A)-AR in ventricular fibrosis and in pathological cardiac remodeling.

Laminopathies are a group of disorders caused by mutations in the LMNA gene that encodes the nuclear lamina proteins, lamin A and lamin C; their pathophysiological basis is unknown. We report that lamin A/C–deficient (Lmna–/–) mice develop rapidly progressive dilated cardiomyopathy (DCM) characterized by left ventricular (LV) dilation and reduced systolic contraction. Isolated Lmna–/– myocytes show reduced shortening with normal baseline and peak amplitude of Ca2+ transients. Lmna–/– LV myocyte nuclei have marked alterations of shape and size with central displacement and fragmentation of heterochromatin; these changes are present but less severe in left atrial nuclei. Electron microscopy of Lmna–/– cardiomyocytes shows disorganization and detachment of desmin filaments from the nuclear surface with progressive disruption of the cytoskeletal desmin network. Alterations in nuclear architecture are associated with defective nuclear function evidenced by decreased SREBP1 import, reduced PPARγ expression, and a lack of hypertrophic gene activation. These findings suggest a model in which the primary pathophysiological mechanism in Lmna–/– mice is defective force transmission resulting from disruption of lamin interactions with the muscle-specific desmin network and loss of cytoskeletal tension. Despite severe DCM, defects in nuclear function prevent Lmna–/– cardiomyocytes from developing compensatory hypertrophy and accelerate disease progression.

The aim of this study was to determine the efficacy of cariporide (a sodium-hydrogen exchanger inhibitor), BMS180448 (a pharmacologic ischemic preconditioning agent), and the combination thereof, as adjuvant therapies for extended cardiac allograft preservation.A porcine model of donor brain death and orthotopic heart transplantation was used. All hearts were arrested and stored for 14 hr in an extracellular preservation solution. Control hearts (CON; n=3) did not receive any additional treatment. Treated hearts received BMS180448 alone (BMS; n=3), cariporide alone (CAR; n=6), or both BMS180448 and cariporide (B+C; n=6). Donors of BMS180448-treated hearts received 2 mg/kg, 15 min before explantation. Donors and recipients of cariporide-treated hearts received 2 mg/kg, 15 min before explantation and reperfusion, respectively.The CON and BMS arms of the study were terminated after three transplantations because initial results in these groups were poor. Significantly, none of the control hearts could be weaned successfully from bypass, whereas all of the treated hearts were weaned successfully (CAR vs. CON and B+C vs. CON: P=0.012). The rate of troponin I release during the first 3 hr after reperfusion was significantly lower in CAR (P=0.0180) and B+C (P=0.0154) recipients than in CON recipients. Mean plasma troponin I levels (microg/mL) 3 hr after reperfusion were as follows: CON 633+/-177, BMS 576+/-110, CAR 346+/-93, and B+C 296+/-97.In this porcine model of extended cardiac allograft preservation, cariporide was more effective than BMS180448 as an adjuvant to our usual preservation solution. There was no additional benefit from the combination of the two therapies.

Background Acute brain death from increased intracranial pressure results in a transient increase in myocardial adenosine and lactate, which indicates that oxygen demand exceeds oxygen delivery during the sympathetic “storm”. The aim of this study was to determine the functional significance of this period of ischemia. Methods Brain death was inflicted on 40 Westran pigs (36.5–68.0 kg) by inflating a 21-ml subdural balloon over 3 minutes. In 38 animals, micromanometry and sonomicrometry were used to obtain left ventricular pressure-volume loops to determine the preload recruitable stroke work (PRSW) relationship. Data files were recorded before and at 15-minute intervals after beginning balloon inflation. Plasma troponin I was measured before and 60 minutes after beginning balloon inflation in the 38 instrumented and 2 non-instrumented animals. Results All animals experienced the classical sympathetic storm. The slope of the PRSW relationship decreased, and the volume-axis intercept shifted to the right 15 minutes after beginning balloon inflation (p < 0.0001). Progressive incremental recovery (leftward shift) occurred between subsequent time points (p ≤ 0.0018). In the instrumented animals, the mean plasma troponin I level increased from 1.4 ± 1.6 μg/liter to 2.8 ± 2.3 μg/liter (p < 0.001). However, troponin I was not detected before or after induction of brain death in the plasma of either non-instrumented animal (p = 0.001). Conclusions The sympathetic storm produced transient contractile dysfunction, consistent with ischemic injury. However, troponin I release reflected surgical instrumentation and not brain death.

Background Acute graft dysfunction caused by ischemia-reperfusion injury is recognized as a major source of morbidity and mortality following adult heart transplantation. The aim of this study was to determine whether treating the donor and recipient with cariporide, an inhibitor of the sodium-hydrogen exchanger, could reduce ischemia-reperfusion injury. Methods. A porcine model of donor brain death, hypothermic ischemic preservation, and orthotopic cardiac transplantation was used. Allografts in both the control group (CON, n=6) and treatment group (CAR, n=6) were arrested and stored for 4 hours in the extracellular crystalloid cardioplegia currently used in the clinical transplantation program at our institution. In addition, both the donor and recipient animals in the CAR group received a single intravenous dose of cariporide (2 mg/kg) 15 minutes before harvesting and reperfusion, respectively. Results. The initial rate of troponin I release was significantly lower in recipients of CAR hearts than in recipients of CON hearts (P=0.020). All hearts were weaned successfully from bypass. More CAR hearts were weaned successfully at the first attempt, at 1 hour post-reperfusion, than CON hearts (6 of 6 vs 3 of 6), but this did not achieve statistical significance. Left ventricular contractility (preload recruitable stroke-work relationship) and left ventricular compliance (end-diastolic pressure-volume relationship) were significantly better preserved in CAR hearts than CON hearts (both P

U74389G (16-desmethyl tirilazad), a 21-aminosteroid or "lazaroid," inhibits lipid peroxidation, which is an important element of ischemia-reperfusion injury. The aim of this study was to determine whether the addition of U74389G to the cardioplegic preservation solution could improve early cardiac allograft function.A porcine model of donor brain death and orthotopic cardiac transplantation was used. Hearts were arrested and preserved for 6 hours in an aspartate-enriched extracellular cardioplegia that had been supplemented with either U74389G and its carrier (n = 7) or the carrier alone (n = 9). Epicardial sonomicrometry and transmyocardial micromanometry were used to obtain pressure-volume loops before and after transplantation. Left ventricular wall volume was measured by volume displacement.A higher proportion of U74389G-treated hearts were weaned successfully from cardiopulmonary bypass, but this difference did not achieve statistical significance (86% [6 of 7] vs 56% [5 of 9]; p = 0.308). In the hearts that were weaned successfully, preservation of left ventricular contractility, as judged by the pre-load recruitable stroke work relationship, was significantly better in the U74389G-treated hearts (p = 0.0271). In contrast, left ventricular compliance, as judged by the end-diastolic pressure-volume relationship, was significantly better preserved in the control group (p0.0001). U74389G-treated hearts developed less myocardial edema, as judged by the post-transplant left ventricular wall volume/baseline steady-state epicardial end-diastolic volume ratio (64 +/- 9% vs 76 +/- 11%; p = 0.045).The benefit obtained from U74389G-supplemented cardioplegic preservation solution was marginal for hearts stored for 6 hours. After longer ischemic times, the benefit may be clearer.

Objective: Paradoxically, it has been reported that after 1.5–4 h of hypothermic ischaemic preservation there is complete recovery of contractile function in canine cardiac allografts, as assessed by the preload recruitable stroke work (PRSW) relationship. This raises questions about the suitability of the canine heart as a model for preservation research and the PRSW relationship as an end-point. The aim of the present study was to evaluate the PRSW relationship as an index of left ventricular contractility in porcine cardiac allografts. Methods: Eighteen orthotopic heart transplants were performed in inbred Westran pigs. Brain death was induced in the donor pigs 1 h prior to explantation. The donor hearts were arrested with extracellular cardioplegia, which was stored in ice prior to administration. On explantation, the donor hearts were immersed in cardioplegia and stored in ice. The donor hearts were subjected to either 4 (IT4, n ¼ 6), 6 (IT6, n ¼ 9) or 14 (IT14, n ¼ 3) h of ischaemia. Post-transplant, all hearts were supported with dobutamine (10 mcg/kg per min). The PRSW relationship was derived from pressure-volume loops obtained by epicardial sonomicrometry and transmyocardial micromanometry. Multiple linear regression was used to describe and compare the PRSW relationship before brain death in the donor and after weaning from bypass in the recipient. Results: Eleven hearts were weaned successfully from cardiopulmonary bypass: IT4 100% (6/6), IT6 56% (5/9) and IT14 0% (0/3) (IT4 versus IT14: P ¼ 0:012). Analysis of the PRSW relationship revealed a reduction in contractility in both the IT4 and IT6 groups (both P , 0:0001), but a greater reduction in the IT6 group (P , 0:0001). Notably, the volume-axis intercept of the PRSW relationship was found to be a better discriminator of post-preservation contractile dysfunction than the slope of the PRSW relationship. Conclusions: The porcine heart’s susceptibility to ischaemic injury makes it ideal for evaluating the effect of different preservation strategies on contractile recovery. The PRSW relationship can be used to evaluate the differences in contractile recovery, though the nature of the effect of ischaemic preservation necessitates analysis by multiple linear regression. q 2002 Elsevier Science B.V. All rights reserved.

Activation of the α 1A -adrenergic receptor (α 1A -AR)/Gq pathway has been implicated as a critical trigger for the development of cardiac hypertrophy. However, direct evidence from in vivo studies is still lacking. To address this issue, transgenic mice with cardiac-targeted overexpression of the α 1A -AR (4- to 170-fold) were generated, using the rodent α-myosin heavy chain promoter. Heterozygous animals displayed marked enhancement of cardiac contractility, evident from increases in dP/dt max (80%, P max /LVP inst (76%, P max :dP/dt min (104%, P P max –end-diastolic volume relationship provided load-independent evidence of a primary increase in contractility. Blood pressure and heart rate were largely unchanged, and there was a small increase in (−)norepinephrine-stimulated, but not basal, phospholipase C activity. Increased contractility was directly related to the level of receptor overexpression and could be completely reversed by acute α 1A - but not β-AR blockade. Despite the robust changes in contractility, transgenic animals displayed no morphological, histological, or echocardiographic evidence of left ventricular hypertrophy. In addition, apart from an increase in atrial natriuretic factor mRNA, expression of other hypertrophy-associated genes was unchanged. To our knowledge, these data provide the first in vivo evidence for an inotropic action of the α 1A -AR.