Paul B. Rosenberg

MicroRNA-Mediated In Vitro and In Vivo Direct Reprogramming of Cardiac Fibroblasts to Cardiomyocytes

Rationale: Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ. Objective: The aim of this study was to develop a strategy that identified and evaluated the potential of specific micro (mi)RNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo. Methods and Results: Using a combinatorial strategy, we identified a combination of miRNAs 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells demonstrated that a single transient transfection of the miRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10-fold on JAK inhibitor I treatment. Importantly, administration of miRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and noncardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. Conclusions: The findings from this study provide proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.

Activation of MEF2 by muscle activity is mediated through a calcineurin‐dependent pathway

Gene expression in skeletal muscles of adult vertebrates is altered profoundly by changing patterns of contractile work. Here we observed that the functional activity of MEF2 transcription factors is stimulated by sustained periods of endurance exercise or motor nerve pacing, as assessed by expression in trans genic mice of a MEF2‐dependent reporter gene (desMEF2‐lacZ). This response is accompanied by transformation of specialized myofiber subtypes, and is blocked either by cyclosporin A, a specific chemical inhibitor of calcineurin, or by forced expression of the endogenous calcineurin inhibitory protein, myocyte‐enriched calcineurin interacting protein 1. Calcineurin removes phosphate groups from MEF2, and augments the potency of the transcriptional activation domain of MEF2 fused to a heterologous DNA binding domain. Across a broad range, the enzymatic activity of calcineurin correlates directly with expression of endogenous genes that are transcriptionally activated by muscle contractions. These results delineate a molecular pathway in which calcineurin and MEF2 participate in the adaptive mechanisms by which skeletal myofibers acquire specialized contractile and metabolic properties as a function of changing patterns of muscle contraction.

STIM1 signalling controls store-operated calcium entry required for development and contractile function in skeletal muscle

It is now well established that stromal interaction molecule 1 (STIM1) is the calcium sensor of endoplasmic reticulum stores required to activate store-operated calcium entry (SOC) channels at the surface of non-excitable cells. However, little is known about STIM1 in excitable cells, such as striated muscle, where the complement of calcium regulatory molecules is rather disparate from that of non-excitable cells. Here, we show that STIM1 is expressed in both myotubes and adult skeletal muscle. Myotubes lacking functional STIM1 fail to show SOC and fatigue rapidly. Moreover, mice lacking functional STIM1 die perinatally from a skeletal myopathy. In addition, STIM1 haploinsufficiency confers a contractile defect only under conditions where rapid refilling of stores would be needed. These findings provide insight into the role of STIM1 in skeletal muscle and suggest that STIM1 has a universal role as an ER/SR calcium sensor in both excitable and non-excitable cells.

β-Arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes

The G protein-coupled receptor kinases (GRKs) and β-arrestins, families of molecules essential to the desensitization of G protein-dependent signaling via seven-transmembrane receptors (7TMRs), have been recently shown to also transduce G protein-independent signals from receptors. However, the physiologic consequences of this G protein-independent, GRK/β-arrestin-dependent signaling are largely unknown. Here, we establish that GRK/β-arrestin-mediated signal transduction via the angiotensin II (ANG) type 1A receptor (AT1AR) results in positive inotropic and lusitropic effects in isolated adult mouse cardiomyocytes. We used the “biased” AT1AR agonist [Sar1, Ile4, Ile8]-angiotensin II (SII), which is unable to stimulate Gαq-mediated signaling, but which has previously been shown to promote β-arrestin interaction with the AT1AR. Cardiomyocytes from WT, but not AT1AR-deficient knockout (KO) mice, exhibited positive inotropic and lusitropic responses to both ANG and SII. Responses of WT cardiomyocytes to ANG were dramatically reduced by protein kinase C (PKC) inhibition, whereas those to SII were unaffected. In contrast, cardiomyocytes from β-arrestin2 KO and GRK6 KO mice failed to respond to SII, but displayed preserved responses to ANG. Cardiomyocytes from GRK2 heterozygous knockout mice (GRK2+/−) exhibited augmented responses to SII in comparison to ANG, whereas those from GRK5 KO mice did not differ from those from WT mice. These findings indicate the existence of independent Gαq/PKC- and GRK6/β-arrestin2-dependent mechanisms by which stimulation of the AT1AR can modulate cardiomyocyte function, and which can be differentially activated by selective receptor ligands. Such ligands may have potential as a novel class of therapeutic agents.

TRPC1 Channels Are Critical for Hypertrophic Signaling in the Heart

Rationale: Cardiac muscle adapts to increase workload by altering cardiomyocyte size and function resulting in cardiac hypertrophy. G protein–coupled receptor signaling is known to govern the hypertrophic response through the regulation of ion channel activity and downstream signaling in failing cardiomyocytes. Objective: Transient receptor potential canonical (TRPC) channels are G protein–coupled receptor operated channels previously implicated in cardiac hypertrophy. Our objective of this study is to better understand how TRPC channels influence cardiomyocyte calcium signaling. Methods and Results: Here, we used whole cell patch clamp of adult cardiomyocytes to show upregulation of a nonselective cation current reminiscent of TRPC channels subjected to pressure overload. This TRPC current corresponds to the increased TRPC channel expression noted in hearts of mice subjected to pressure overload. Importantly, we show that mice lacking TRPC1 channels are missing this putative TRPC current. Moreover, Trpc1−/− mice fail to manifest evidence of maladaptive cardiac hypertrophy and maintain preserved cardiac function when subjected to hemodynamic stress and neurohormonal excess. In addition, we provide a mechanistic basis for the protection conferred to Trpc1−/− mice as mechanosensitive signaling through calcineurin/NFAT, mTOR and Akt is altered in Trpc1−/− mice. Conclusions: From these studies, we suggest that TRPC1 channels are critical for the adaptation to biomechanical stress and TRPC dysregulation leads to maladaptive cardiac hypertrophy and failure.

Exercise Can Prevent and Reverse the Severity of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common form of sudden death in young competitive athletes. However, exercise has also been shown to be beneficial in the setting of other cardiac diseases. We examined the ability of voluntary exercise to prevent or reverse the phenotypes of a murine model of HCM harboring a mutant myosin heavy chain (MyHC). No differences in voluntary cage wheel performance between nontransgenic (NTG) and HCM male mice were seen. Exercise prevented fibrosis, myocyte disarray, and induction of “hypertrophic” markers including NFAT activity when initiated before established HCM pathology. If initiated in older HCM animals with documented disease, exercise reversed myocyte disarray (but not fibrosis) and “hypertrophic” marker induction. In addition, exercise returned the increased levels of phosphorylated GSK-3&bgr; to those of NTG and decreased levels of phosphorylated CREB in HCM mice to normal levels. Exercise in HCM mice also favorably impacted components of the apoptotic signaling pathway, including Bcl-2 (an inhibitor of apoptosis) and procaspase-9 (an effector of apoptosis) expression, and caspase-3 activity. Remarkably, there were no differences in mortality between exercised NTG and HCM mice. Thus, not only was exercise not harmful but also it was able to prevent and even reverse established cardiac disease phenotypes in this HCM model.

Comparison of impedance cardiography with invasive hemodynamic measurements in patients with heart failure secondary to ischemic or nonischemic cardiomyopathy.

Right-sided cardiac catheterization is frequently performed in patients with heart failure despite potential complications including arrhythmias, right ventricular or pulmonary artery perforation, pulmonary infarction, infection, 1 and possibly increased mortality. 2,3 A noninvasive measure of cardiac output and left ventricular fi lling pressures would therefore be desirable. Impedance cardiography via a commercially available device (BioZ.com, CardioDynamics International Corporation, San Diego, California) may permit such noninvasive measurement of hemodynamics by assessing the change in impedance of an alternating current applied across the thorax to determine stroke volume, cardiac output, and thoracic fl uid content. 4 However, it is not known how well its as

TRPC6 enhances angiotensin II-induced albuminuria.

Mutations in the canonical transient receptor potential cation channel 6 (TRPC6) are responsible for familial forms of adult onset focal segmental glomerulosclerosis (FSGS). The mechanisms by which TRPC6 mutations cause kidney disease are not well understood. We used TRPC6-deficient mice to examine the function of TRPC6 in the kidney. We found that adult TRPC6-deficient mice had BP and albumin excretion rates similar to wild-type animals. Glomerular histomorphology revealed no abnormalities on both light and electron microscopy. To determine whether the absence of TRPC6 would alter susceptibility to hypertension and renal injury, we infused mice with angiotensin II continuously for 28 days. Although both groups developed similar levels of hypertension, TRPC6-deficient mice had significantly less albuminuria, especially during the early phase of the infusion; this suggested that TRPC6 adversely influences the glomerular filter. We used whole-cell patch-clamp recording to measure cell-membrane currents in primary cultures of podocytes from both wild-type and TRPC6-deficient mice. In podocytes from wild-type mice, angiotensin II and a direct activator of TRPC6 both augmented cell-membrane currents; TRPC6 deficiency abrogated these increases in current magnitude. Our findings suggest that TRPC6 promotes albuminuria, perhaps by promoting angiotensin II-dependent increases in Ca(2+), suggesting that TRPC6 blockade may be therapeutically beneficial in proteinuric kidney disease.

MicroRNA Induced Cardiac Reprogramming In Vivo Evidence for Mature Cardiac Myocytes and Improved Cardiac Function

Rationale: A major goal for the treatment of heart tissue damaged by cardiac injury is to develop strategies for restoring healthy heart muscle through the regeneration and repair of damaged myocardium. We recently demonstrated that administration of a specific combination of microRNAs (miR combo) into the infarcted myocardium leads to direct in vivo reprogramming of noncardiac myocytes to cardiac myocytes. However, the biological and functional consequences of such reprogramming are not yet known. Objective: The aim of this study was to determine whether noncardiac myocytes directly reprogrammed using miRNAs in vivo develop into mature functional cardiac myocytes in situ, and whether reprogramming leads to improvement of cardiac function. Methods and Results: We subjected fibroblast-specific protein 1-Cre mice/tandem dimer Tomato (tdTomato) mice to cardiac injury by permanent ligation of the left anterior descending coronary artery and injected lentiviruses encoding miR combo or a control nontargeting miRNA. miR combo significantly increased the number of reprogramming events in vivo. Five to 6 weeks after injury, morphological and physiological properties of tdTomato− and tdTomato+ cardiac myocyte–like cells were analyzed ex vivo. tdTomato+ cells expressed cardiac myocyte markers, sarcomeric organization, excitation–contraction coupling, and action potentials characteristic of mature ventricular cardiac myocytes (tdTomato− cells). Reprogramming was associated with improvement of cardiac function, as analyzed by serial echocardiography. There was a time delayed and progressive improvement in fractional shortening and other measures of ventricular function, indicating that miR combo promotes functional recovery of damaged myocardium. Conclusions: The findings from this study further validate the potential use of miRNA-mediated reprogramming as a therapeutic approach to promote cardiac regeneration after myocardial injury.Rationale: A major goal for the treatment of heart tissue damaged by cardiac injury is to develop strategies for restoring healthy heart muscle through the regeneration and repair of damaged myocardium. We recently demonstrated that administration of a specific combination of microRNAs (miR combo) into the infarcted myocardium leads to direct in vivo reprogramming of noncardiac myocytes to cardiac myocytes. However, the biological and functional consequences of such reprogramming are not yet known. Objective: The aim of this study was to determine whether noncardiac myocytes directly reprogrammed using miRNAs in vivo develop into mature functional cardiac myocytes in situ, and whether reprogramming leads to improvement of cardiac function. Methods and Results: We subjected fibroblast-specific protein 1-Cre mice/tandem dimer Tomato (tdTomato) mice to cardiac injury by permanent ligation of the left anterior descending coronary artery and injected lentiviruses encoding miR combo or a control nontargeting miRNA. miR combo significantly increased the number of reprogramming events in vivo. Five to 6 weeks after injury, morphological and physiological properties of tdTomato− and tdTomato+ cardiac myocyte–like cells were analyzed ex vivo. tdTomato+ cells expressed cardiac myocyte markers, sarcomeric organization, excitation–contraction coupling, and action potentials characteristic of mature ventricular cardiac myocytes (tdTomato− cells). Reprogramming was associated with improvement of cardiac function, as analyzed by serial echocardiography. There was a time delayed and progressive improvement in fractional shortening and other measures of ventricular function, indicating that miR combo promotes functional recovery of damaged myocardium. Conclusions: The findings from this study further validate the potential use of miRNA-mediated reprogramming as a therapeutic approach to promote cardiac regeneration after myocardial injury. # Novelty and Significance {#article-title-18}

Mice Lacking Homer 1 Exhibit a Skeletal Myopathy Characterized by Abnormal Transient Receptor Potential Channel Activity

ABSTRACT Transient receptor potential (TRP) channels are nonselective cation channels, several of which are expressed in striated muscle. Because the scaffolding protein Homer 1 has been implicated in TRP channel regulation, we hypothesized that Homer proteins play a significant role in skeletal muscle function. Mice lacking Homer 1 exhibited a myopathy characterized by decreased muscle fiber cross-sectional area and decreased skeletal muscle force generation. Homer 1 knockout myotubes displayed increased basal current density and spontaneous cation influx. This spontaneous cation influx in Homer 1 knockout myotubes was blocked by reexpression of Homer 1b, but not Homer 1a, and by gene silencing of TRPC1. Moreover, diminished Homer 1 expression in mouse models of Duchennes muscular dystrophy suggests that loss of Homer 1 scaffolding of TRP channels may contribute to the increased stretch-activated channel activity observed in mdx myofibers. These findings provide direct evidence that Homer 1 functions as an important scaffold for TRP channels and regulates mechanotransduction in skeletal muscle.