MCU-independent Ca2+ uptake mediates mitochondrial Ca2+ overload and necrotic cell death in a mouse model of Duchenne muscular dystrophy.

Mitochondrial Ca2+ overload can mediate mitochondria-dependent cell death, a major contributor to several human diseases. Indeed, Duchenne muscular dystrophy (MD) is driven by dysfunctional Ca2+ influx across the sarcolemma that causes mitochondrial Ca2+ overload, organelle rupture, and muscle necrosis. The mitochondrial Ca2+ uniporter (MCU) complex is the primary characterized mechanism for acute mitochondrial Ca2+ uptake. One strategy for preventing mitochondrial Ca2+ overload is deletion of the Mcu gene, the pore forming subunit of the MCU-complex. Conversely, enhanced MCU-complex Ca2+ uptake is achieved by deleting the inhibitory Mcub gene. Here we show that myofiber-specific Mcu deletion was not protective in a mouse model of Duchenne MD. Specifically, Mcu gene deletion did not reduce muscle histopathology, did not improve muscle function, and did not prevent mitochondrial Ca2+ overload. Moreover, myofiber specific Mcub gene deletion did not augment Duchenne MD muscle pathology. Interestingly, we observed MCU-independent Ca2+ uptake in dystrophic mitochondria that was sufficient to drive mitochondrial permeability transition pore (MPTP) activation and skeletal muscle necrosis, and this same type of activity was observed in heart, liver, and brain mitochondria. These results demonstrate that mitochondria possess an uncharacterized MCU-independent Ca2+ uptake mechanism that is sufficient to drive MPTP-dependent necrosis in MD in vivo.

Col1a2-Deleted Mice Have Defective Type I Collagen and Secondary Reactive Cardiac Fibrosis with Altered Hypertrophic Dynamics.

RATIONALE: The adult cardiac extracellular matrix (ECM) is largely comprised of type I collagen. In addition to serving as the primary structural support component of the cardiac ECM, type I collagen also provides an organizational platform for other ECM proteins, matricellular proteins, and signaling components that impact cellular stress sensing in vivo. OBJECTIVE: Here we investigated how the content and integrity of type I collagen affect cardiac structure function and response to injury. METHODS AND RESULTS: We generated and characterized Col1a2-/- mice using standard gene targeting. Col1a2-/- mice were viable, although by young adulthood their hearts showed alterations in ECM mechanical properties, as well as an unanticipated activation of cardiac fibroblasts and induction of a progressive fibrotic response. This included augmented TGFß activity, increases in fibroblast number, and progressive cardiac hypertrophy, with reduced functional performance by 9 months of age. Col1a2-loxP-targeted mice were also generated and crossed with the tamoxifen-inducible Postn-MerCreMer mice to delete the Col1a2 gene in myofibroblasts with pressure overload injury. Interestingly, while germline Col1a2-/- mice showed gradual pathologic hypertrophy and fibrosis with aging, the acute deletion of Col1a2 from activated adult myofibroblasts showed a loss of total collagen deposition with acute cardiac injury and an acute reduction in pressure overload-induce cardiac hypertrophy. However, this reduction in hypertrophy due to myofibroblast-specific Col1a2 deletion was lost after 2 and 6 weeks of pressure overload, as fibrotic deposition accumulated. CONCLUSIONS: Defective type I collagen in the heart alters the structural integrity of the ECM and leads to cardiomyopathy in adulthood, with fibroblast expansion, activation, and alternate fibrotic ECM deposition. However, acute inhibition of type I collagen production can have an anti-fibrotic and anti-hypertrophic effect.

ANT-dependent MPTP underlies necrotic myofiber death in muscular dystrophy.

Mitochondrial permeability transition pore (MPTP) formation contributes to ischemia-reperfusion injury in the heart and several degenerative diseases, including muscular dystrophy (MD). MD is a family of genetic disorders characterized by progressive muscle necrosis and premature death. It has been proposed that the MPTP has two molecular components, the adenine nucleotide translocase (ANT) family of proteins and an unknown component that requires the chaperone cyclophilin D (CypD) to activate. This model was examined in vivo by deleting the gene encoding ANT1 (Slc25a4) or CypD (Ppif) in a δ-sarcoglycan (Sgcd) gene-deleted mouse model of MD, revealing that dystrophic mice lacking Slc25a4 were partially protected from cell death and MD pathology. Dystrophic mice lacking both Slc25a4 and Ppif together were almost completely protected from necrotic cell death and MD disease. This study provides direct evidence that ANT1 and CypD are required MPTP components governing in vivo cell death, suggesting a previously unrecognized therapeutic approach in MD and other necrotic diseases.

A human FLII gene variant alters sarcomeric actin thin filament length and predisposes to cardiomyopathy.

To better understand the genetic basis of heart disease, we identified a variant in the Flightless-I homolog (FLII) gene that generates a R1243H missense change and predisposes to cardiac remodeling across multiple previous human genome-wide association studies (GWAS). Since this gene is of unknown function in the mammalian heart we generated gain- and loss-of-function genetically altered mice, as well as knock-in mice with the syntenic R1245H amino acid substitution, which showed that Flii protein binds the sarcomeric actin thin filament and influences its length. Deletion of Flii from the heart, or mice with the R1245H amino acid substitution, show cardiomyopathy due to shortening of the actin thin filaments. Mechanistically, Flii is a known actin binding protein that we show associates with tropomodulin-1 (TMOD1) to regulate sarcomere thin filament length. Indeed, overexpression of leiomodin-2 in the heart, which lengthens the actin-containing thin filaments, partially rescued disease due to heart-specific deletion of Flii. Collectively, the identified FLII human variant likely increases cardiomyopathy risk through an alteration in sarcomere structure and associated contractile dynamics, like other sarcomere gene-based familial cardiomyopathies.

An enhancer-based gene-therapy strategy for spatiotemporal control of cargoes during tissue repair.

The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals.

Seroprevalence of SARS-CoV-2 infection in Cincinnati Ohio USA from August to December 2020.

The world is currently in a pandemic of COVID-19 (Coronavirus disease-2019) caused by a novel positive-sense, single-stranded RNA ß-coronavirus referred to as SARS-CoV-2. Here we investigated rates of SARS-CoV-2 infection in the greater Cincinnati, Ohio, USA metropolitan area from August 13 to December 8, 2020, just prior to initiation of the national vaccination program. Examination of 9,550 adult blood donor volunteers for serum IgG antibody positivity against the SARS-CoV-2 Spike protein showed an overall prevalence of 8.40%, measured as 7.56% in the first 58 days and 9.24% in the last 58 days, and 12.86% in December 2020, which we extrapolated to ~20% as of March, 2021. Males and females showed similar rates of past infection, and rates among Hispanic or Latinos, African Americans and Whites were also investigated. Donors under 30 years of age had the highest rates of past infection, while those over 60 had the lowest. Geographic analysis showed higher rates of infectivity on the West side of Cincinnati compared with the East side (split by I-75) and the lowest rates in the adjoining region of Kentucky (across the Ohio river). These results in regional seroprevalence will help inform efforts to best achieve herd immunity in conjunction with the national vaccination campaign.

Nanoparticle Delivery of STAT3 Alleviates Pulmonary Hypertension in a Mouse Model of Alveolar Capillary Dysplasia.

BACKGROUND: Pulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy. METHODS: We used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1WT/S52F mice and determine its effects on PH and RV hypertrophy. RESULTS: Foxf1WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling. CONCLUSIONS: Nanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.

A specialized population of Periostin-expressing cardiac fibroblasts contributes to postnatal cardiomyocyte maturation and innervation.

During the postnatal period in mammals, the cardiac muscle transitions from hyperplasic to hypertrophic growth, the extracellular matrix (ECM) undergoes remodeling, and the heart loses regenerative capacity. While ECM maturation and crosstalk between cardiac fibroblasts (CFs) and cardiomyocytes (CMs) have been implicated in neonatal heart development, not much is known about specialized fibroblast heterogeneity and function in the early postnatal period. In order to better understand CF functions in heart maturation and postnatal cardiomyocyte cell-cycle arrest, we have performed gene expression profiling and ablation of postnatal CF populations. Fibroblast lineages expressing Tcf21 or Periostin were traced in transgenic GFP reporter mice, and their biological functions and transitions during the postnatal period were examined in sorted cells using RNA sequencing. Highly proliferative Periostin (Postn)+ lineage CFs were found from postnatal day 1 (P1) to P11 but were not detected at P30, due to a repression of Postn gene expression. This population was less abundant and transcriptionally different from Tcf21+ resident CFs. The specialized Postn+ population preferentially expresses genes related to cell proliferation and neuronal development, while Tcf21+ CFs differentially express genes related to ECM maturation at P7 and immune crosstalk at P30. Ablation of the Postn+ CFs from P0 to P6 led to altered cardiac sympathetic nerve patterning and a reduction in binucleation and hypertrophic growth with increased fetal troponin (TroponinI1) expression in CM. Thus, postnatal CFs are heterogeneous and include a transient proliferative Postn+ population required for cardiac nerve development and cardiomyocyte maturation soon after birth.

An acute immune response underlies the benefit of cardiac stem cell therapy.

Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day1,2, despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biological effect3. The rationale for these cell therapy trials is derived from animal studies that show a modest but reproducible improvement in cardiac function in models of cardiac ischaemic injury4,5. Here we examine the mechanistic basis for cell therapy in mice after ischaemia-reperfusion injury, and find that-although heart function is enhanced-it is not associated with the production of new cardiomyocytes. Cell therapy improved heart function through an acute sterile immune response characterized by the temporal and regional induction of CCR2+ and CX3CR1+ macrophages. Intracardiac injection of two distinct types of adult stem cells, cells killed by freezing and thawing or a chemical inducer of the innate immune response all induced a similar regional accumulation of CCR2+ and CX3CR1+ macrophages, and provided functional rejuvenation to the heart after ischaemia-reperfusion injury. This selective macrophage response altered the activity of cardiac fibroblasts, reduced the extracellular matrix content in the border zone and enhanced the mechanical properties of the injured area. The functional benefit of cardiac cell therapy is thus due to an acute inflammatory-based wound-healing response that rejuvenates the infarcted area of the heart.

The EYA3 tyrosine phosphatase activity promotes pulmonary vascular remodeling in pulmonary arterial hypertension.

In pulmonary hypertension vascular remodeling leads to narrowing of distal pulmonary arterioles and increased pulmonary vascular resistance. Vascular remodeling is promoted by the survival and proliferation of pulmonary arterial vascular cells in a DNA-damaging, hostile microenvironment. Here we report that levels of Eyes Absent 3 (EYA3) are elevated in pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension and that EYA3 tyrosine phosphatase activity promotes the survival of these cells under DNA-damaging conditions. Transgenic mice harboring an inactivating mutation in the EYA3 tyrosine phosphatase domain are significantly protected from vascular remodeling. Pharmacological inhibition of the EYA3 tyrosine phosphatase activity substantially reverses vascular remodeling in a rat model of angio-obliterative pulmonary hypertension. Together these observations establish EYA3 as a disease-modifying target whose function in the pathophysiology of pulmonary arterial hypertension can be targeted by available inhibitors.

Defective Flux of Thrombospondin-4 through the Secretory Pathway Impairs Cardiomyocyte Membrane Stability and Causes Cardiomyopathy.

Thrombospondins are stress-inducible secreted glycoproteins with critical functions in tissue injury and healing. Thrombospondin-4 (Thbs4) is protective in cardiac and skeletal muscle, where it activates an adaptive endoplasmic reticulum (ER) stress response, induces expansion of the ER, and enhances sarcolemmal stability. However, it is unclear if Thbs4 has these protective functions from within the cell, from the extracellular matrix, or from the secretion process itself. In this study, we generated transgenic mice with cardiac cell-specific overexpression of a secretion-defective mutant of Thbs4 to evaluate its exclusive intracellular and secretion-dependent functions. Like wild-type Thbs4, the secretion-defective mutant upregulates the adaptive ER stress response and expands the ER and intracellular vesicles in cardiomyocytes. However, only the secretion-defective Thbs4 mutant produces cardiomyopathy with sarcolemmal weakness and rupture that is associated with reduced adhesion-forming glycoproteins in the membrane. Similarly, deletion of Thbs4 in the mdx mouse model of Duchenne muscular dystrophy enhances cardiomyocyte membrane instability and cardiomyopathy. Finally, overexpression of the secretion-defective Thbs4 mutant in Drosophila, but not wild-type Thbs4, impaired muscle function and sarcomere alignment. These results suggest that transit through the secretory pathway is required for Thbs4 to augment sarcolemmal stability, while ER stress induction and vesicular expansion mediated by Thbs4 are exclusively intracellular processes.

TGFBI functions similar to periostin but is uniquely dispensable during cardiac injury.

Extracellular matrix production and accumulation stabilize the heart under normal conditions as well as form a protective scar after myocardial infarction injury, although excessive extracellular matrix accumulation with long-standing heart disease is pathological. In the current study we investigate the role of the matricellular protein, transforming growth factor beta-induced (TGFBI), which is induced in various forms of heart disease. Additionally, we sought to understand whether TGFBI is functionally redundant to its closely related family member periostin, which is also induced in the diseased heart. Surgical models of myocardial infarction and cardiac pressure overload were used in mice with genetic loss of Postn and/or Tgfbi to examine the roles of these genes during the fibrotic response. Additionally, cardiac-specific TGFBI transgenic mice were generated and analyzed. We observed that deletion of Tgfbi did not alter cardiac disease after myocardial infarction in contrast to greater ventricular wall rupture in Postn gene-deleted mice. Moreover, Tgfbi and Postn double gene-deleted mice showed a similar post-myocardial infarction disease phenotype as Postn-deleted mice. Over-expression of TGFBI in the hearts of mice had a similar effect as previously shown in mice with periostin over-expression. Thus, TGFBI and periostin act similarly in the heart in affecting fibrosis and disease responsiveness, although TGFBI is not seemingly necessary in the heart after myocardial infarction injury and is fully compensated by the more prominently expressed effector periostin.

Mitsugumin 29 regulates t-tubule architecture in the failing heart.

Transverse tubules (t-tubules) are uniquely-adapted membrane invaginations in cardiac myocytes that facilitate the synchronous release of Ca2+ from internal stores and subsequent myofilament contraction, although these structures become disorganized and rarefied in heart failure. We previously observed that mitsugumin 29 (Mg29), an important t-tubule organizing protein in skeletal muscle, was induced in the mouse heart for the first time during dilated cardiomyopathy with heart failure. Here we generated cardiac-specific transgenic mice expressing Mg29 to model this observed induction in the failing heart. Interestingly, expression of Mg29 in the hearts of Csrp3 null mice (encoding muscle LIM protein, MLP) partially restored t-tubule structure and preserved cardiac function as measured by invasive hemodynamics, without altering Ca2+ spark frequency. Conversely, gene-deleted mice lacking both Mg29 and MLP protein showed a further reduction in t-tubule organization and accelerated heart failure. Thus, induction of Mg29 in the failing heart is a compensatory response that directly counteracts the well-characterized loss of t-tubule complexity and reduced expression of anchoring proteins such as junctophilin-2 (Jph2) that normally occur in this disease. Moreover, preservation of t-tubule structure by Mg29 induction significantly increases the function of the failing heart.

Caveolae-localized L-type Ca2+ channels do not contribute to function or hypertrophic signalling in the mouse heart.

AIMS: L-type Ca2+ channels (LTCCs) in adult cardiomyocytes are localized to t-tubules where they initiate excitation-contraction coupling. Our recent work has shown that a subpopulation of LTCCs found at the surface sarcolemma in caveolae of adult feline cardiomyocytes can also generate a Ca2+ microdomain that activates nuclear factor of activated T-cells signaling and cardiac hypertrophy, although the relevance of this paradigm to hypertrophy regulation in vivo has not been examined. METHODS AND RESULTS: Here we generated heart-specific transgenic mice with a putative caveolae-targeted LTCC activator protein that was ineffective in initiating or enhancing cardiac hypertrophy in vivo. We also generated transgenic mice with cardiac-specific overexpression of a putative caveolae-targeted inhibitor of LTCCs, and while this protein inhibited caveolae-localized LTCCs without effects on global Ca2+ handling, it similarly had no effect on cardiac hypertrophy in vivo. Cardiac hypertrophy was elicited by pressure overload for 2 or 12 weeks or with neurohumoral agonist infusion. Caveolae-specific LTCC activator or inhibitor transgenic mice showed no greater change in nuclear factor of activated T-cells activity after 2 weeks of pressure overload stimulation compared with control mice. CONCLUSION: Our results indicate that LTCCs in the caveolae microdomain do not affect cardiac function and are not necessary for the regulation of hypertrophic signaling in the adult mouse heart.

Nemo-Like Kinase (NLK) Is a Pathological Signaling Effector in the Mouse Heart.

Nemo-like kinase (NLK) is an evolutionary conserved serine/threonine protein kinase implicated in development, proliferation and apoptosis regulation. Here we identified NLK as a gene product induced in the hearts of mice subjected to pressure overload or myocardial infarction injury, suggesting a potential regulatory role with pathological stimulation to this organ. To examine the potential functional consequences of increased NLK levels, cardiac-specific transgenic mice with inducible expression of this gene product were generated, as well as cardiac-specific Nlk gene-deleted mice. NLK transgenic mice demonstrated baseline cardiac hypertrophy, dilation, interstitial fibrosis, apoptosis and progression towards heart failure in response to two surgery-induced cardiac disease models. In contrast, cardiac-specific deletion of Nlk from the heart, achieved by crossing a Nlk-loxP allele containing mouse with either a mouse containing a ß-myosin heavy chain promoter driven Cre transgene or a tamoxifen inducible α-myosin heavy chain promoter containing transgene driving a MerCreMer cDNA, protected the mice from cardiac dysfunction following pathological stimuli. Mechanistically, NLK interacted with multiple proteins including the transcription factor Stat1, which was significantly increased in the hearts of NLK transgenic mice. These results indicate that NLK is a pathological effector in the heart.

DUSP8 Regulates Cardiac Ventricular Remodeling by Altering ERK1/2 Signaling.

RATIONALE: Mitogen-activated protein kinase (MAPK) signaling regulates the growth response of the adult myocardium in response to increased cardiac workload or pathological insults. The dual-specificity phosphatases (DUSPs) are critical effectors, which dephosphorylate the MAPKs to control the basal tone, amplitude, and duration of MAPK signaling. OBJECTIVE: To examine DUSP8 as a regulator of MAPK signaling in the heart and its impact on ventricular and cardiac myocyte growth dynamics. METHODS AND RESULTS: Dusp8 gene-deleted mice and transgenic mice with inducible expression of DUSP8 in the heart were used here to investigate how this MAPK-phosphatase might regulate intracellular signaling and cardiac growth dynamics in vivo. Dusp8 gene-deleted mice were mildly hypercontractile at baseline with a cardiac phenotype of concentric ventricular remodeling, which protected them from progressing towards heart failure in 2 surgery-induced disease models. Cardiac-specific overexpression of DUSP8 produced spontaneous eccentric remodeling and ventricular dilation with heart failure. At the cellular level, adult cardiac myocytes from Dusp8 gene-deleted mice were thicker and shorter, whereas DUSP8 overexpression promoted cardiac myocyte lengthening with a loss of thickness. Mechanistically, activation of extracellular signal-regulated kinases 1/2 were selectively increased in Dusp8 gene-deleted hearts at baseline and following acute pathological stress stimulation, whereas p38 MAPK and c-Jun N-terminal kinases were mostly unaffected. CONCLUSIONS: These results indicate that DUSP8 controls basal and acute stress-induced extracellular signal-regulated kinases 1/2 signaling in adult cardiac myocytes that then alters the length-width growth dynamics of individual cardiac myocytes, which further alters contractility, ventricular remodeling, and disease susceptibility.

A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy.

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

Cardiac-specific deletion of protein phosphatase 1ß promotes increased myofilament protein phosphorylation and contractile alterations.

There are 3 protein phosphatase 1 (PP1) catalytic isoforms (α, ß and γ) encoded within the mammalian genome. These 3 gene products share ~90% amino acid homology within their catalytic domains but each has unique N- and C-termini that likely underlie distinctive subcellular localization or functionality. In this study, we assessed the effect associated with the loss of each PP1 isoform in the heart using a conditional Cre-loxP targeting approach in mice. Ppp1ca-loxP, Ppp1cb-loxP and Ppp1cc-loxP alleles were crossed with either an Nkx2.5-Cre knock-in containing allele for early embryonic deletion or a tamoxifen inducible α-myosin heavy chain (αMHC)-MerCreMer transgene for adult and cardiac-specific deletion. We determined that while deletion of Ppp1ca (PP1α) or Ppp1cc (PP1γ) had little effect on the whole heart, deletion of Ppp1cb (PP1ß) resulted in concentric remodeling of the heart, interstitial fibrosis and contractile dysregulation, using either the embryonic or adult-specific Cre-expressing alleles. However, myocytes isolated from Ppp1cb deleted hearts surprisingly showed enhanced contractility. Mechanistically we found that deletion of any of the 3 PP1 gene-encoding isoforms had no effect on phosphorylation of phospholamban, nor were Ca(2+) handling dynamics altered in adult myocytes from Ppp1cb deleted hearts. However, the loss of Ppp1cb from the heart, but not Ppp1ca or Ppp1cc, resulted in elevated phosphorylation of myofilament proteins such as myosin light chain 2 and cardiac myosin binding protein C, consistent with an enriched localization profile of this isoform to the sarcomeres. These results suggest a unique functional role for the PP1ß isoform in affecting cardiac contractile function.

STIM1 elevation in the heart results in aberrant Ca²âº 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 6weeks of age, while mice surviving past 12weeks 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.

The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart.

In the heart, augmented Ca(2+) fluxing drives contractility and ATP generation through mitochondrial Ca(2+) loading. Pathologic mitochondrial Ca(2+) overload with ischemic injury triggers mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte death. Mitochondrial Ca(2+) uptake is primarily mediated by the mitochondrial Ca(2+) uniporter (MCU). Here, we generated mice with adult and cardiomyocyte-specific deletion of Mcu, which produced mitochondria refractory to acute Ca(2+) uptake, with impaired ATP production, and inhibited MPTP opening upon acute Ca(2+) challenge. Mice lacking Mcu in the adult heart were also protected from acute ischemia-reperfusion injury. However, resting/basal mitochondrial Ca(2+) levels were normal in hearts of Mcu-deleted mice, and mitochondria lacking MCU eventually loaded with Ca(2+) after stress stimulation. Indeed, Mcu-deleted mice were unable to immediately sprint on a treadmill unless warmed up for 30 min. Hence, MCU is a dedicated regulator of short-term mitochondrial Ca(2+) loading underlying a "fight-or-flight" response that acutely matches cardiac workload with ATP production.